Macrophage stimulating 1 receptor (MST1r) variants and uses thereof

Abstract

To provide methods of treating patients having inflammatory bowel disease (IBD) or primary sclerosing cholangitis (PSC).SOLUTION: The present invention provides a method comprising administering to the patient an agonist of the Macrophage Stimulating 1 (MST1) / Macrophage Stimulating 1 Receptor (MST1R) pathway.SELECTED DRAWING: None

Description

Technical Field

[0001] Reference to the Sequence Listing This application includes a Sequence Listing that was electronically submitted as a text file named 18923802902SEQ, created on Aug. 8, 2020, and having a size of 201 kilobytes. The Sequence Listing is hereby incorporated by reference into this specification.

[0002] Technical Field The present disclosure provides methods for treating patients having inflammatory bowel disease (IBD) or primary sclerosing cholangitis (PSC).

Background Art

[0003] IBD is a group of inflammatory conditions of the colon and small intestine that cause over 50,000 deaths per year. The cause of IBD is complex and factors can include diet, genetics, and the composition of an individual's gastrointestinal microbiota. Medical treatment generally depends on factors specific to the individual.

[0004] Crohn's disease (CD) and ulcerative colitis (UC) are among the most common forms of IBD. Both CD and UC are inflammatory diseases, but UC is located in the colon and CD can affect any part of the gastrointestinal tract from the mouth to the anus. Neither CD nor UC is currently medically curable, and current treatments range from surgical removal of part of the intestine to administration of anti-inflammatory and / or immunosuppressive drugs. Unfortunately, current treatments for CD and UC are often ineffective and can cause significant side effects.

[0005] Primary sclerosing cholangitis (PSC) was initially defined as a chronic cholestatic liver disease characterized by fibrotic inflammation of segments of the extrahepatic bile ducts. PSC results in progressive stricture or occlusion of the bile duct lumen and progression of secondary biliary cirrhosis, with complications of portal hypertension, liver failure, and cholangiocarcinoma. It is an idiopathic disorder characterized by inflammation and obstruction of both intrahepatic and extrahepatic bile ducts. Furthermore, more than approximately two-thirds of all PSC patients are complicated by inflammatory bowel disease (IBD). However, the relationship between these two diseases remains undefined. Bile duct cells account for 3% to 5% of the liver cell population and cover a complex network of interconnected ducts within the liver, known as the intrahepatic biliary tree. One of the pathological conditions that occurs in both intrahepatic and extrahepatic bile ducts is PSC. Medical treatment of PSC includes corticosteroids, antibiotics, immunosuppressive agents, and choleretics, either alone or in combination. Usually, all outcomes are disappointing, and the median number of years from diagnosis to death is 10 years in the absence of liver transplantation. Thus, new methods and compositions for the treatment of IBD and / or PSC are still needed.

[0006] MST1 encodes macrophage stimulating protein (MSP; alternatively referred to as the MST1 protein), a liver-secreted protein that binds to its associated receptor, MST1R (alternatively referred to as Recepteur d’Origine Nantais, or RON), which is expressed in macrophages, Kupffer cells, and intestinal epithelial cells, among other cell types. MST1 / MST1R signaling promotes macrophage polarization, cell chemotaxis, and epithelial wound repair. Previous genome-wide association studies (GWAS) in patients with PSC and IBD, as well as healthy controls, have identified a common missense variant (3:49684099:G:A; rs3197999) in MST1 that is associated with an increased risk of PSC (OR 1.3, p = 2e-26) and IBD (OR 1.2, p = 1e-47) in published meta-GWAS. Functional studies have shown that 3:49684099:G:A rs3197999 is associated with a decrease in MST1 protein levels in human serum. Collectively, these data indicate that decreased MST1 is associated with an increased risk of PSC and IBD.

[0007] MST1R is a cell surface receptor for MST1 that has tyrosine kinase activity. The mature form of this protein is a disulfide-linked heterodimer of alpha and beta subunits that is generated by proteolytic cleavage of a single-chain precursor. MST1R functions as a tyrosine kinase that transmits signals from the extracellular matrix into the cytoplasm by binding to the MST1 ligand. To date, MST1R has not been independently associated with IBD or PSC in genetic association studies. SUMMARY OF THE INVENTION

[0008] The present disclosure provides a method of treating a patient having IBD, the method comprising administering to the patient an agonist of the MST1 / MST1R pathway. The present disclosure also provides a method of treating a patient having PSC, the method comprising administering to the patient an agonist of the MST1 / MST1R pathway.

[0009] The present disclosure also provides, in some embodiments, such methods in which the presence or absence of MST1 and / or MST1R variant nucleic acid molecules or variant polypeptides associated with an increased risk of developing IBD and / or PSC in a biological sample from a patient is detected.

[0010] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain some of the principles of the present disclosure. This patent file or application file contains at least one drawing created in color. Copies of this patent or patent application publication with color drawings will be provided by the United States Patent and Trademark Office upon request and payment of the required fees.

Brief Description of the Drawings

[0011]

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Best Mode for Carrying Out the Invention

[0012] Various terms related to aspects of the present disclosure are used throughout this specification and the entire scope of the claims. Unless otherwise specified, such terms shall be given their ordinary meanings in the relevant technical field. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0013] Unless otherwise clearly stated, no method or aspect described in this specification is intended to be construed as requiring its steps to be performed in a particular order. Thus, unless specifically recited in the claims or the description as limiting the steps of a method claim to a particular order, no inference of order is intended in any respect. This is true for any possible implicit interpretive criteria, including logical matters regarding the sequence of steps or workflows, general meanings derived from grammatical construction or punctuation, or the number or type of aspects described in this specification.

[0014] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "subject" and "patient" are used interchangeably. A subject can include any animal, including mammals. Mammals include, but are not limited to, domestic animals (e.g., horses, cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), and non-human primates. In some embodiments, the subject is a human.

[0015] As used herein, "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide", or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded or multi-stranded. One strand of a nucleic acid also refers to its complement.

[0016] As used herein, the term "comprising" may, in certain embodiments, be replaced, at the option of the user, with "consisting of" or "consisting essentially of". As used herein, the phrase "corresponding to" or its grammatical variants, when used in the context of a particular amino acid or nucleotide sequence or position number, refers to the number of the reference sequence when the particular amino acid or nucleotide sequence is compared to a predetermined reference sequence (e.g., the reference sequence in the present invention is a nucleic acid molecule or polypeptide of (wild-type) MST1R). In other words, the number or position of a residue (e.g., amino acid or nucleotide) of a particular polymer is assigned based on a reference sequence, rather than by the actual position number of that residue in the particular amino acid or nucleotide sequence. For example, a particular amino acid or nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue identity between the two sequences. In these cases, even if gaps are present, the numbering of residues in the particular amino acid or nucleotide sequence is based on the reference sequence to which the sequence is aligned. There are various computational algorithms that can be used to perform sequence alignment to identify nucleotide or amino acid positions in one polymer molecule that correspond to nucleotide or amino acid positions in another polymer molecule. For example, the NCBI BLAST algorithm (Altschul Sequence alignment can be performed by using, for example, the ClustalW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116) or Nucleic Acids Res., 1997, 25, 3389-3402) et al. However, the sequences can also be aligned manually.

[0017] According to the present disclosure, it has been observed that the set burden of certain variations in MST1R is associated with the risk of developing IBD or PSC. Variants in the MST1R gene or protein are not thought to be associated with IBD or PSC in genome-wide or exome-wide association studies. Thus, a human having an MST1R change associated with IBD or PSC can be treated such that IBD or PSC is suppressed, its symptoms are reduced, and / or the onset of symptoms is inhibited. Also, a human having IBD or PSC can be thought to be treatable with a molecule that promotes MST1 / MST1R signaling.

[0018] For the purposes of the present disclosure, any particular human can be classified as having any combination of the following MST1 and / or MST1R genotypes: i) MST1 and / or MST1R reference; ii) heterozygosity for MST1 and / or MST1R variant nucleic acid molecules associated with an increased risk of developing IBD and / or PSC, and iii) homozygosity for MST1 and / or MST1R variant nucleic acid molecules associated with an increased risk of developing IBD and / or PSC. A human who does not have a copy of an MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is an MST1 and / or MST1R reference. A human who has a single copy of an MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is heterozygous for the MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC. A human who has any two copies of an MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is homozygous for the MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC.

[0019] MST1 and / or MST1R variant nucleic acid molecules associated with an increased risk of developing IBD and / or PSC are any MST1 and / or MST1R nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, or cDNA molecules generated from mRNA molecules) that encode an MST1 and / or MST1R polypeptide having a partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function (collectively, "predicted loss of function" variant nucleic acid molecules). A human having an MST1 and / or MST1R polypeptide with a partial loss of function (or predicted partial loss of function) is hypomorphic for MST1 and / or MST1R. The predicted loss of function variant nucleic acid molecules of MST1 and / or MST1R can be any one or more of the variant nucleic acid molecules described herein.

[0020] MST1 and / or MST1R variant nucleic acid molecules associated with an increased risk of developing IBD and / or PSC are also any MST1 and / or MST1R deleterious missense variant nucleic acid molecules. The MST1 and / or MST1R deleterious missense variant nucleic acid molecules can be any one or more of the variant nucleic acid molecules described herein.

[0021] In the case of a human subject or patient who is genotyped or determined to be heterozygous or homozygous for an MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC, such a human subject or patient is associated with having an increased odds of developing IBD and / or PSC.

[0022] In any of the embodiments described herein, an MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC can be any MST1 and / or MST1R nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule generated from an mRNA molecule, etc.) that encodes an MST1 and / or MST1R polypeptide having a partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. In any of the embodiments described herein, an MST1 and / or MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC can also be any MST1 and / or MST1R missense variant nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule generated from an mRNA molecule, etc.).

[0023] The nucleotide sequence of the MST1R reference genomic nucleic acid molecule is shown in SEQ ID NO: 1, which is 16,636 nucleotides in length. The first nucleotide described in SEQ ID NO: 1 corresponds to the nucleotide at position 49,903,637 on chromosome 3 (see hg38_knownGene_ENSG00000164078.12).

[0024] (Using the human genome reference build GRch38): There are a number of variant genomic nucleic acid molecules of MST1R, including but not limited to 3:49903264:CG:C, 3:49903084:C:T, 3:49890026:G:T, 3:49902417:A:G, 3:49902560:A:T, and 3:49903387:G:T. Thus, for example, using the reference genomic nucleotide sequence (SEQ ID NO: 1) as the base sequence (having the first nucleotide listed therein designated as position 49,903,637), the first-listed variant (3:49903264:CG:C) has a CG dinucleotide replaced with cytosine (referred to as the "variant nucleotide") at position 49,903,264 (referred to as the "variant position") that effectively deletes guanine. In another example, the second-listed variant (3:49903084:C:T) has cytosine replaced with thymine (referred to as the "variant nucleotide") at position 49,903,084 (referred to as the "variant position"). Any of these predicted loss-of-function variant genomic nucleic acid molecules of MST1R can be detected by any of the methods described herein.

[0025] The nucleotide sequences of the MST1R reference mRNA molecules generated via alternative splicing are shown in SEQ ID NOs: 2-10. The variant nucleotides at their respective variant positions for the variant genomic nucleic acid molecules described herein also have corresponding variant nucleotides at their respective variant positions for the variant mRNA molecules based on the MST1R reference mRNA sequences according to SEQ ID NOs: 2-10. Any of these MST1R variant mRNA molecules can be detected by any of the methods described herein.

[0026] The nucleotide sequences of the MST1R reference cDNA molecules are shown in SEQ ID NOs: 11 to 19. The variant nucleotides at their respective variant positions for the variant genomic nucleic acid molecules described herein also have corresponding variant nucleotides at their respective variant positions for the variant cDNA molecules based on the MST1R reference cDNA sequence according to SEQ ID NOs: 11 to 19. Any of these MST1R variant cDNA molecules can be detected by any of the methods described herein.

[0027] The amino acid sequences of the MST1R reference polypeptide isoforms are shown in SEQ ID NOs: 20 to 28. Using the translated nucleotide sequence of either the MST1R mRNA or cDNA molecule, the MST1R variant polypeptide has corresponding translated variant amino acids at the variant positions (codons). Any of these MST1R variant polypeptides can be detected by any of the methods described herein.

[0028] The nucleotide sequence of the MST1 reference genomic nucleic acid molecule is shown in SEQ ID NO: 29, which is 5,107 nucleotides in length. The first nucleotide shown in SEQ ID NO: 29 corresponds to the nucleotide at position 49,689,053 on chromosome 3 according to the GRCh38 / hg38 human genome assembly (see hg38_knownGene_ENSG00000449682.2).

[0029] The nucleotide sequence of the rs3197999 variant genomic nucleic acid molecule is shown in SEQ ID NO: 30. Compared with the reference genomic nucleic acid molecule sequence (SEQ ID NO: 29), the nucleotide sequence of the rs3197999 variant genomic nucleic acid molecule (SEQ ID NO: 30) contains thymine instead of cytosine at position 4,955. Compared with the reference genomic nucleic acid molecule sequence (SEQ ID NO: 29), the nucleotide sequence of the rs3197999 variant genomic nucleic acid molecule (SEQ ID NO: 30) contains the TGC codon instead of the CGC codon at positions 4,955 - 4,957. This predicted loss-of-function variant genomic nucleic acid molecule of MST1 can be detected by any of the methods described herein.

[0030] The nucleotide sequence of the rs142690032 variant genomic nucleic acid molecule is shown in SEQ ID NO: 37. Compared with the reference genomic nucleic acid molecule sequence (SEQ ID NO: 29), the nucleotide sequence of the rs142690032 variant genomic nucleic acid molecule (SEQ ID NO: 37) contains thymine instead of cytosine at position 4,675. Compared with the reference genomic nucleic acid molecule sequence (SEQ ID NO: 29), the nucleotide sequence of the rs142690032 variant genomic nucleic acid molecule (SEQ ID NO: 37) contains the ACT codon instead of the ACC codon at positions 4,673 - 4,675. This predicted loss-of-function variant genomic nucleic acid molecule of MST1 can be detected by any of the methods described herein.

[0031] The nucleotide sequence of the MST1 reference mRNA molecule is shown in SEQ ID NO: 31. The nucleotide sequence of the rs3197999 mRNA molecule is shown in SEQ ID NO: 32. Compared with the reference mRNA molecule sequence (SEQ ID NO: 31), the nucleotide sequence of the rs3197999 variant mRNA molecule (SEQ ID NO: 32) contains uracil instead of cytosine at position 2,469. Compared with the reference mRNA molecule sequence (SEQ ID NO: 31), the nucleotide sequence of the rs3197999 variant mRNA molecule (SEQ ID NO: 32) contains the UGC codon instead of the CGC codon at positions 2,469 - 2,471. This predicted loss-of-function variant mRNA molecule of MST1 can be detected by any of the methods described herein.

[0032] The nucleotide sequence of the rs142690032 variant mRNA molecule is shown in SEQ ID NO: 38. Compared with the reference mRNA molecule sequence (SEQ ID NO: 31), the nucleotide sequence of the rs142690032 variant mRNA molecule (SEQ ID NO: 38) contains uracil instead of cytosine at position 2,313. Compared with the reference mRNA molecule sequence (SEQ ID NO: 31), the nucleotide sequence of the rs142690032 variant mRNA molecule (SEQ ID NO: 38) contains the ACU codon instead of the ACC codon at positions 2,311 to 2,313. This predicted loss-of-function variant genomic nucleic acid molecule of MST1 can be detected by any of the methods described herein.

[0033] The nucleotide sequence of the MST1 reference cDNA molecule is shown in SEQ ID NO: 33. The nucleotide sequence of the rs3197999 cDNA molecule is shown in SEQ ID NO: 34. Compared with the reference cDNA molecule sequence (SEQ ID NO: 33), the nucleotide sequence of the rs3197999 variant cDNA molecule (SEQ ID NO: 34) contains thymine instead of cytosine at position 2,469. Compared with the reference cDNA molecule sequence (SEQ ID NO: 33), the nucleotide sequence of the rs3197999 variant cDNA molecule (SEQ ID NO: 34) contains the TGC codon instead of the CGC codon at positions 2,469 to 2,471. This predicted loss-of-function variant cDNA molecule of MST1 can be detected by any of the methods described herein.

[0034] The nucleotide sequence of the rs142690032 variant cDNA molecule is shown in SEQ ID NO: 39. Compared with the reference cDNA molecule sequence (SEQ ID NO: 33), the nucleotide sequence of the rs142690032 variant cDNA molecule (SEQ ID NO: 39) contains thymine instead of cytosine at position 2,313. Compared with the reference cDNA molecule sequence (SEQ ID NO: 33), the nucleotide sequence of the rs142690032 variant cDNA molecule (SEQ ID NO: 39) contains the ACT codon instead of the ACC codon at positions 2,311 - 2,313. This predicted loss-of-function variant cDNA molecule of MST1 can be detected by any of the methods described herein.

[0035] The amino acid sequence of the reference MST1 polypeptide is shown in SEQ ID NO: 35 and is 725 amino acids in length. The amino acid sequence of the rs3197999 variant MST1 polypeptide is shown in SEQ ID NO: 36 and is 725 amino acids in length. The SNP in the underlying nucleic acid molecule results in a substitution of cytosine for arginine at amino acid position 703 of the reference MST1 polypeptide (SEQ ID NO: 35). This rs3197999 variant MST1 polypeptide is referred to herein as the Arg703Cys MST1 polypeptide.

[0036] The amino acid sequence of the rs142690032 variant MST1 polypeptide is shown in SEQ ID NO: 40 and is 650 amino acids in length. The SNP in the underlying nucleic acid molecule results in the acquisition of a stop codon (replacing arginine) at the position corresponding to position 651 according to SEQ ID NO: 35. This results in the expression of a variant MST1 polypeptide truncated at the position corresponding to position 650 according to SEQ ID NO: 35. This rs142690032 variant MST1 polypeptide is referred to herein as the Arg651STOP MST1 polypeptide. This variant MST1 polypeptide does not contain amino acids at positions corresponding to positions 651 - 725 of the reference MST1 polypeptide (SEQ ID NO: 35).

[0037] In any of the embodiments described herein, the variant MST1 nucleic acid molecule or polypeptide can be or can encode Arg703Cys or Arg651STOP. In any of the embodiments described herein, the variant MST1 nucleic acid molecule or polypeptide can be or can encode Arg703Cys. In any of the embodiments described herein, the variant MST1 nucleic acid molecule or polypeptide can be or can encode Arg651STOP.

[0038] The nucleotide sequences and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and the three-letter notation for amino acids. Nucleotide sequences follow the standard convention of starting at the 5' end of the sequence and proceeding towards the 3' end (i.e., from left to right in each row). Only one strand of each nucleotide sequence is shown, but any reference to the shown strand is understood to include the complementary strand. Amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding towards the carboxy terminus (i.e., from left to right in each row).

[0039] The present disclosure provides a method of treating a patient having IBD, the method comprising administering to the patient an agonist of the MST1 / MST1R pathway. In any of the embodiments described herein, the IBD is ulcerative colitis (UC) or Crohn's disease (CD). In any of the embodiments described herein, the CD is jejunoileitis, ileitis, ileocolitis, or Crohn's colitis, or any combination thereof. In any of the embodiments described herein, the UC is ulcerative proctitis, left-sided colitis, or extensive colitis, or any combination thereof.

[0040] The symptoms of Crohn's disease include, but are not limited to, frequent and / or recurrent diarrhea, rectal bleeding, unexplained weight loss, fever, abdominal pain, cramps, fatigue, a feeling of low energy, and loss of appetite. The symptoms of ulcerative proctitis include, but are not limited to, rectal bleeding, rectal pain, and urgency of bowel movements. The symptoms of left-sided colitis include, but are not limited to, loss of appetite, weight loss, hemorrhagic diarrhea, and pain on the left side of the abdomen. The symptoms of extensive colitis include, but are not limited to, loss of appetite, hemorrhagic diarrhea, abdominal pain, and weight loss.

[0041] The present disclosure also provides a method of treating a patient having PSC, the method comprising administering to the patient an agonist of the MST1 / MST1R pathway. In any of the embodiments described herein, the PSC is early-stage PSC (e.g., stage 1 according to Ludwig et al., Hepatology, 1981, 1, 632-640). In any of the embodiments described herein, the PSC is late-stage PSC (e.g., stage 4 according to Ludwig et al., Hepatology, 1981, 1, 632-640). In any of the embodiments described herein, the PSC is at any disease stage (e.g., stage 4 according to Ludwig et al., Hepatology, 1981, 1, 632-640). In some embodiments, the human subject is female. In some embodiments, the human subject is male.

[0042] The symptoms of PSC include, but are not limited to, jaundice, non-specific upper abdominal pain, fatigue, itching, right upper abdomen, pain and jaundice of the skin and mucous membranes, and scleral (obstructive jaundice). Further symptoms include a general feeling of unwellness (malaise); abdominal pain, particularly in the right upper abdomen; nausea; dark urine; light-colored stools; unexpected weight loss, and / or abnormal enlargement of the liver (hepatomegaly) and / or abnormal enlargement of the spleen (splenomegaly).

[0043] In any of the embodiments described herein, an agonist of the MST1 / MST1R pathway is an MST1 agonist or an MST1R agonist. In some embodiments, the MST1 agonist and / or MST1R agonist comprises a protein, a nucleic acid molecule, or a small molecule. In some embodiments, the protein is an antibody against MST1 or MST1R.

[0044] In some embodiments, the MST1 agonist is a protein such as recombinant MST1. In some embodiments, the recombinant MST1 is a fusion protein (e.g., Mst1-Fc) comprising MST1 or a fragment thereof fused to a heterologous protein, such as an antibody or a fragment thereof, such as an Fc fragment. In some embodiments, the MST1 agonist is kereberin, recombinant tumor necrosis factor receptor-associated factor 2 (TRAF2), or curcumin. In some embodiments, the MST1 agonist is a protease selected from hepatocyte growth factor activator (HGFA), matriptase, hepsin, TMPRSS11D (human airway trypsin-like protease; HAT), coagulation factor XIIa, coagulation factor Xia, and kallikrein.

[0045] In some embodiments, the MST1R agonist is recombinant macrophage scavenger receptor 1 (MSR1), recombinant hepatocyte growth factor-like protein (HGFL), or recombinant androgen receptor (AR). In some embodiments, the MST1R agonist is an antibody selected from mAb Zt / g4, mAb Zt / c1, mAb Zt / f2, mAb Zt / 64, mAb 3F12, mAb B9, and mAb 1G4.

[0046] In any of the embodiments described herein, patients having IBD and / or PSC may be treated with additional therapeutic agents. Examples of therapeutic agents for treating or suppressing IBD include aminosalicylates (e.g., COLAZAL™ ( balsalazide), ASACOL™, APRISO™, LIALDA™, and PENTASA™ ( mesalamine), DIPENTUM™ ( olsalazine), and AZULFIDINE™ ( sulfasalazine)); corticosteroids; immunomodulators (e.g., IMURAN™ ( azathioprine), RHEUMATREX™ ( methotrexate), and PURINETHOL™ ( 6-mercaptopurine (6-MP)); and antibodies (e.g., REMICADE™ ( infliximab), RENFLEXIS™ ( infliximab-abda), INFLECTRA™ ( infliximab-dyyb), HUMIRA™ ( adalimumab), AMJEVITA™ ( adalimumab-atto), CIMZIA™ ( certolizumab), TYSABRI™ ( natalizumab), ENTYVIO™ ( vedolizumab), STELARA™ ( ustekinumab), SIMPONI™ and SIMPONI™ ARIA™ ( golimumab)), as well as recombinant MST1, or any combination thereof, but are not limited thereto. Examples of therapeutic agents for treating or suppressing IBD include aminosalicylates (e.g., balsalazide, mesalamine, olsalazine, and sulfasalazine); corticosteroids; immunomodulators (e.g., azathioprine, methotrexate, and 6-mercaptopurine (6-MP)); and antibodies (e.g., infliximab, infliximab-abda, infliximab-dyyb, adalimumab, adalimumab-atto, certolizumab, natalizumab, vedolizumab, ustekinumab, golimumab), as well as recombinant MST1, or any combination thereof, but are not limited thereto. Examples of therapeutic agents for treating or suppressing PSC include agents for reducing itching and jaundice, antibiotics for treating infections, immunosuppressants, gallbladder stimulants, and vitamin supplements, or any combination thereof, but are not limited thereto.

[0047] Administration of a therapeutic agent for treating or suppressing IBD or PSC can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. The repeated administration can be at the same dose or at different doses. The administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. For example, according to a particular dosing regimen, a patient may receive treatment for a long period, such as 6 months, 1 year, or more.

[0048] Administration of a therapeutic agent for treating or suppressing IBD or PSC can be carried out by any suitable route including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical composition for administration is preferably sterile, substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a dosage for single administration). The pharmaceutical composition can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, additive, or adjuvant is compatible with the other components of the formulation and is not substantially harmful to the recipient.

[0049] As used herein, the terms “treating,” “treatment,” “treat,” “preventing,” “prevention,” and “prevent” each refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect. In some embodiments, the therapeutic effect includes a decrease / reduction in IBD, PSC, or both, a decrease / reduction in the severity of IBD, PSC, or both (e.g., a decrease or inhibition in the progression of IBD, PSC, or both), a decrease / reduction in symptoms and IBD-related effects, PSC-related effects, or both, a delay in the onset of symptoms and IBD-related effects, PSC-related effects, or both, a decrease in the severity of IBD-related effects, PSC-related effects, or both, a decrease in the severity of acute flares, a decrease in the number of symptoms and IBD-related effects, PSC-related effects, or both, a decrease in the latency period of symptoms and IBD-related effects, PSC-related effects, or both, an improvement in symptoms and IBD-related effects, PSC-related effects, or both, a decrease in secondary symptoms, a decrease in secondary infections, prevention of relapse of IBD, PSC, or both, a decrease in the number or frequency of relapse flares, an increase in the latency period between flares presenting symptoms, an increase in the time to prolonged progression, an acceleration of recovery, and / or an increase in the effectiveness of alternative treatments or a decrease in their resistance, including one or more of the foregoing. The prophylactic effect can include complete or partial avoidance / suppression or delay (e.g., complete or partial avoidance / suppression or delay) of the onset / progression of IBD, PSC, or both after implementation of a treatment protocol. Treatment of IBD, PSC, or both includes treatment of patients already diagnosed as having any form of IBD, PSC, or both at any clinical stage or symptom, delay in the onset or progression or exacerbation or worsening of symptoms or signs of IBD, PSC, or both, and / or prevention and / or reduction in the severity of IBD, PSC, or both.

[0050] In any of the embodiments described herein, the method may further comprise detecting the presence or absence of MST1 and / or MST1R variant nucleic acid molecules or variant polypeptides associated with an increased risk of developing IBD and / or PSC in a biological sample from a patient. It should be understood that gene sequences within a population, and mRNA molecules encoded by such genes, may differ due to polymorphisms such as single nucleotide polymorphisms. The sequences provided herein for the MST1 and / or MST1R variant nucleic acid molecules disclosed herein are merely exemplary sequences. Other sequences for the MST1 and / or MST1R variant nucleic acid molecules are possible.

[0051] MST1 and / or MST1R variant nucleic acid molecules or variant polypeptides associated with an increased risk of developing IBD and / or PSC can be any predicted loss-of-function variant of any MST1 and / or MST1R or MST1 and / or MST1R missense variants, such as any of those described herein. For example, in any of the embodiments described herein, an MST1 variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is Arg703Cys or Arg651STOP. In some embodiments, an MST1 variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is Arg703Cys. In some embodiments, an MST1 variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is Arg651STOP. Also, in any of the embodiments described herein, an MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC can be 3:49903264:CG:C, 3:49903084:C:T, 3:49890026:G:T, 3:49902417:A:G, 3:49902560:A:T, or 3:49903387:G:T, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule. In any of the embodiments described herein, an MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is 3:49903264:CG:C, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule. In any of the embodiments described herein, an MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is 3:49903084:C:T, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule. In any of the embodiments described herein, an MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is 3:49890026:G:T, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule.In any of the embodiments described herein, the MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is 3:49902417:A:G, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule. In any of the embodiments described herein, the MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is 3:49902560:A:T, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule. In any of the embodiments described herein, the MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC can be 3:49903387:G:T, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule.

[0052] In some embodiments, detecting the presence or absence of MST1 and / or MST1R variant nucleic acid molecules or variant polypeptides associated with an increased risk of developing IBD and / or PSC comprises determining whether a patient has an MST1 and / or MST1R variant genomic nucleic acid molecule associated with an increased risk of developing IBD and / or PSC, an MST1 and / or MST1R variant mRNA molecule associated with an increased risk of developing IBD and / or PSC, an MST1 and / or MST1R variant cDNA molecule generated from the mRNA molecule, and / or an MST1 and / or MST1R variant polypeptide associated with an increased risk of developing IBD and / or PSC. In some embodiments, such determination is made by obtaining or having obtained a biological sample from the patient and performing or having performed an assay on the biological sample to determine whether the patient has an MST1 and / or MST1R variant nucleic acid molecule or variant polypeptide associated with an increased risk of developing IBD and / or PSC. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0053] Determining whether a human has an MST1 and / or MST1R variant nucleic acid molecule or an MST1 and / or MST1R variant polypeptide associated with an increased risk of developing IBD and / or PSC in a biological sample can be performed by any of the methods described herein. In some embodiments, the detecting, determining, or assay is performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present within cells obtained from a human subject. In some embodiments, the assay is a genotyping assay for the nucleic acid molecule. In some embodiments, the assay is an immunoassay for the polypeptide.

[0054] A biological sample can be derived from any cell, tissue, or biological fluid from a subject. The sample can include a bone marrow sample, a tumor biopsy, a fine needle aspiration biopsy, or any clinically relevant tissue such as a body fluid sample, e.g., blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some cases, the sample includes an oral swab. In some embodiments, the biological sample includes a cell lysate. Such methods can further include obtaining a biological sample from a subject. The samples used in the methods disclosed herein will vary based on the assay format, the nature of the detection method, and the tissue, cells, or extract used as the sample. The biological sample can be processed differently depending on the assay employed. For example, when detecting any MST1 and / or MST1R variant nucleic acid molecule, a pretreatment designed to isolate or enrich the sample for genomic DNA can be used. Various known techniques can be used for this purpose. When detecting the level of any MST1 and / or MST1R variant mRNA, different techniques can be used to enrich the biological sample having the mRNA. Various methods can be used to detect the presence or level of the mRNA, or the presence of a particular variant genomic DNA locus.

[0055] In some embodiments, the method may further comprise determining the aggregate burden of patients having MST1 and / or MST1R variant genomic nucleic acid molecules associated with an increased risk of developing IBD and / or PSC, MST1 and / or MST1R variant mRNA molecules associated with an increased risk of developing IBD and / or PSC, MST1 and / or MST1R variant cDNA molecules generated from the mRNA molecules, and / or MST1 and / or MST1R variant polypeptides associated with an increased risk of developing IBD and / or PSC. The aggregate burden is the sum of all rare variants in the MSTR1 gene, which is performed in an association study with IBD. The results of the association study suggest that rare loss-of-function and missense variants of MST1R are associated with increased odds of IBD. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0056] In some embodiments, the detecting, determining, or assaying step comprises sequencing at least a portion of the nucleotide sequence of the MST1 and / or MST1R nucleic acid molecule in the biological sample. The portion to be sequenced comprises positions corresponding to predicted loss-of-function variant positions. If a variant nucleotide at a predicted loss-of-function variant position is detected, the MST1 and / or MST1R nucleic acid molecule in the biological sample is a predicted loss-of-function variant nucleic acid molecule of MST1 and / or MST1R. The predicted loss-of-function variant positions within any particular MST1 and / or MST1R nucleic acid molecule are one or more positions of a variant nucleotide sequence that is different as compared to the nucleotide sequence of the corresponding reference nucleic acid molecule. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0057] In some embodiments, the detecting, determining, or assay comprises contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an MST1 and / or MST1R nucleic acid molecule proximal to the predicted loss-of-function variant position, extending the primer through at least the predicted loss-of-function variant position, and determining whether the extension product of the primer contains variant nucleotides at the predicted loss-of-function variant position. In some embodiments, the detecting, determining, or assay comprises sequencing the entire nucleic acid molecule. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0058] In some embodiments, the detecting, determining, or assay comprises sequencing at least a portion of the nucleotide sequence of an MST1 and / or MST1R nucleic acid molecule in a biological sample. The portion to be sequenced includes positions corresponding to missense variant positions. If a variant nucleotide at a missense variant position is detected, the MST1 and / or MST1R nucleic acid molecule in the biological sample is an MST1 and / or MST1R missense variant nucleic acid molecule. The missense variant position in any particular MST1 and / or MST1R nucleic acid molecule is one or more positions of a variant nucleotide sequence that is different as compared to the nucleotide sequence of the corresponding reference nucleic acid molecule. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0059] In some embodiments, the detecting, determining, or assay comprises contacting a biological sample with a primer that hybridizes to a portion of the nucleotide sequence of an MST1 and / or MST1R nucleic acid molecule proximate to a missense variant position, extending the primer through at least the missense variant position, and determining whether the extension product of the primer contains a variant nucleotide at the missense variant position. In some embodiments, the detecting, determining, or assay comprises sequencing the entire nucleic acid molecule. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0060] In some embodiments, the assay comprises contacting a biological sample with a primer, e.g., a change-specific primer that specifically hybridizes under stringent conditions to an MST1 or MST1R variant genomic sequence, variant mRNA sequence, or variant cDNA sequence and does not specifically hybridize to the corresponding MST1 or MST1R reference sequence. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0061] In some embodiments, only MST1 and / or MST1R genomic nucleic acid molecules are analyzed. In some embodiments, only MST1 and / or MST1R mRNA are analyzed. In some embodiments, only MST1 and / or MST1R cDNA obtained from MST1 and / or MST1R mRNA are analyzed. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0062] Exemplary examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. In some embodiments, the assay includes RNA sequencing (RNA-Seq). In some embodiments, the assay also includes reverse transcribing mRNA to cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0063] In some embodiments, the detecting, determining, or assaying comprises amplifying at least a portion of an MST1 and / or MST1R nucleic acid molecule, wherein a portion thereof comprises a predicted loss-of-function variant position, amplifying, labeling the amplified nucleic acid molecule with a detectable label, contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the predicted loss-of-function variant position, contacting, and detecting the detectable label. In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0064] In some embodiments, the detecting, determining, or assaying comprises contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a predicted loss-of-function variant position, contacting, and detecting the detectable label. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0065] In some embodiments, the detecting, determining, or assay comprises amplifying at least a portion of an MST1 and / or MST1R nucleic acid molecule, wherein a portion thereof comprises a missense variant position, amplifying, labeling the amplified nucleic acid molecule with a detectable label, contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes to the missense variant position under stringent conditions, contacting, and detecting the detectable label. In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0066] In some embodiments, the detecting, determining, or assay comprises contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes to the missense variant position under stringent conditions, contacting, and detecting the detectable label. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0067] The change-specific probes or change-specific primers described herein are complementary to, hybridize with, or specifically hybridize with a predicted loss-of-function variant nucleic acid molecule of MST1 or MST1R, or a missense variant nucleic acid molecule of MST1 or MST1R, or its complement, and contain a nucleic acid sequence. In some embodiments, the change-specific probe or change-specific primer contains or consists 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, or at least about 50 nucleotides. In some embodiments, the change-specific probe or change-specific primer contains or consists of at least 15 nucleotides. In some embodiments, the change-specific probe or change-specific primer contains or consists of at least 15 nucleotides to at least about 35 nucleotides. In some embodiments, the change-specific probe or change-specific primer hybridizes with a predicted loss-of-function variant genomic nucleic acid molecule of MST1 or MST1R, a predicted loss-of-function variant mRNA molecule of MST1R, and / or a predicted loss-of-function variant cDNA molecule of MST1R under stringent conditions. In some embodiments, the change-specific probe or change-specific primer hybridizes with a missense variant genomic nucleic acid molecule of MST1 or MST1R, a missense variant mRNA molecule of MST1R, and / or a missense variant cDNA molecule of MST1R under stringent conditions. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP.In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0068] The change-specific polymerase chain reaction technique can be used to detect mutations such as SNPs in nucleic acid sequences. Since DNA polymerase does not elongate when there is a mismatch with the template, change-specific primers can be used.

[0069] In some embodiments, the assay comprises contacting a biological sample with a probe, e.g., a change-specific probe that specifically hybridizes to an MST1 or MST1R variant genomic sequence, variant mRNA sequence, or variant cDNA sequence under stringent conditions and does not specifically hybridize to the corresponding MST1 or MST1R reference sequence, and determining whether hybridization has occurred. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0070] In some embodiments, the method utilizes probes and primers of a nucleotide length sufficient to specifically detect and / or identify a polynucleotide that binds to a target nucleotide sequence and comprises an MST1 or MST1R variant genomic nucleic acid molecule, variant mRNA molecule, or variant cDNA molecule. 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 an optimal detection method, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under high stringency hybridization conditions. Unlike the target nucleotide sequence, probes that retain the ability to specifically detect and / or identify the target nucleotide sequence may be designed by conventional methods, but the probes and primers may have complete nucleotide sequence identity of contiguous nucleotides within the target nucleotide sequence. The probes and primers can have a sequence identity or complementarity of 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% with the nucleotide sequence of the target nucleic acid molecule.

[0071] In some embodiments, labeled primers or probes directed against purified DNA, amplified DNA, and fixed cell preparations (fluorescent in situ hybridization (FISH)) can be used for detection. In some methods, the target nucleic acid molecule may be amplified before or simultaneously with detection. Exemplary examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification methods, and thermophilic SDA (tSDA).

[0072] In hybridization techniques, stringent conditions can be employed so that a probe or primer can hybridize specifically to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions hybridizes to its target sequence to a detectably higher degree, e.g., at least 2-fold, at least 3-fold, at least 4-fold, or more, e.g., more than 10-fold background, than to other non-target sequences. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0073] Suitable stringency conditions that promote DNA hybridization, e.g., 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by a wash in 2× SSC at 50°C, are known and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection are at pH 7.0 - 8.3, with a salt concentration of less than about 1.5 M Na+ ions, typically about 0.01 - 1.0 M Na+ ion concentration (or other salts), and a temperature of at least about 30°C for short probes (e.g., 10 - 50 nucleotides, etc.) and at least about 60°C for longer probes (e.g., greater than 50 nucleotides, etc.). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Optionally, the wash buffer can contain about 0.1% - about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 - about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0074] In some embodiments, detecting the presence of a human MST1 and / or MST1R variant polypeptide comprises performing an assay on a sample obtained from a human subject to determine whether the MST1 and / or MST1R polypeptide in the subject has one or more variations that cause the polypeptide to have a loss of function (partial or complete) or a predicted loss of function (partial or complete), or is generated from a missense variant nucleic acid molecule. In some embodiments, the assay comprises sequencing at least a portion of the MST1 and / or MST1R polypeptide that includes the variant position. In some embodiments, the step of detecting comprises sequencing the entire polypeptide. Identification of variant amino acids at variant positions of the MST1 and / or MST1R polypeptide indicates that the MST1 and / or MST1R polypeptide is a predicted loss-of-function polypeptide of MST1 and / or MST1R or is generated from a missense variant nucleic acid molecule. In some embodiments, the assay comprises an immunoassay for detecting the presence of the variant polypeptide. Detection of variant amino acids at variant positions of the MST1 or MST1R polypeptide indicates that the MST1 or MST1R polypeptide is a variant MST1 or MST1R polypeptide. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0075] The probes and / or primers (including variant-specific probes and variant-specific primers) described herein comprise or consist of from about 15 to about 100, or from about 15 to about 35 nucleotides. In some embodiments, the variant-specific probes and variant-specific primers comprise DNA. In some embodiments, the variant-specific probes and variant-specific primers comprise RNA. In some embodiments, the probes and primers (including variant-specific probes and variant-specific primers) described herein have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or its complement. In some embodiments, the probes and primers (including variant-specific probes and variant-specific primers) specifically hybridize under stringent conditions to any of the nucleic acid molecules disclosed herein. As used in the context of the present disclosure, "specifically hybridize" means that a probe or primer (including variant-specific probes and variant-specific primers) does not hybridize to the nucleic acid sequence encoding the MST1R reference genomic nucleic acid molecule, MST1R reference mRNA molecule, and / or MST1R reference cDNA molecule. In some embodiments, the probe (e.g., a variant-specific probe, etc.) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0076] The present disclosure also provides a method of identifying a human subject having an increased risk of developing IBD. The method includes determining or having determined whether the subject has any one or more of the predicted loss-of-function variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. The method may also include determining or having determined whether the subject has any one or more of the missense variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. If the subject has a predicted loss-of-function variant nucleic acid molecule of MST1R, or a missense variant nucleic acid molecule of MST1R, or a polypeptide generated therefrom, the subject has an increased risk of developing IBD. In some embodiments, if a human subject has been identified as having an increased risk of developing IBD, the human subject is further treated with a therapeutic agent and / or an MST1 / MST1R pathway agonist for treating or suppressing IBD as described herein. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0077] The present disclosure also provides a method of identifying a human subject having an increased risk of developing PSC. The method includes determining or having determined whether the subject has any one or more of the predicted loss-of-function variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. The method may also include determining or having determined whether the subject has any one or more of the missense variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. If the subject has a predicted loss-of-function variant nucleic acid molecule of MST1R, or a missense variant nucleic acid molecule of MST1R, or a polypeptide generated therefrom, the subject has an increased risk of developing PSC. In some embodiments, if a human subject has been identified as having an increased risk of developing PSC, the human subject is further treated with a therapeutic agent and / or an MST1 / MST1R pathway agonist that treats or suppresses PSC as described herein. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0078] The present disclosure also provides a method for diagnosing IBD in a human subject. The method includes determining or having determined whether the subject has any one or more of the predicted loss-of-function variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. The method may also include determining or having determined whether the subject has any one or more of the missense variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. If the subject has a predicted loss-of-function variant nucleic acid molecule of MST1R, or a missense variant nucleic acid molecule of MST1R, or a polypeptide generated therefrom, and has one or more symptoms of IBD, the subject is diagnosed as having IBD. In some embodiments, if a human subject has been identified as having IBD, the human subject is further treated with a therapeutic agent and / or an MST1 / MST1R pathway agonist described herein for treating or suppressing IBD. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0079] The present disclosure also provides a method for diagnosing PSC in a human subject. The method includes determining or having determined whether the subject has any one or more of the predicted loss-of-function variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. The method may also include determining or having determined whether the subject has any one or more of the missense variant nucleic acid molecules of MST1R described herein or polypeptides generated therefrom. If the subject has a predicted loss-of-function variant nucleic acid molecule of MST1R, or a missense variant nucleic acid molecule of MST1R, or a polypeptide generated therefrom, and has one or more symptoms of IBD, the subject is diagnosed as having PSC. In some embodiments, if a human subject has been identified as having PSC, the human subject is further treated with a therapeutic agent and / or an MST1 / MST1R pathway agonist that treats or suppresses PSC as described herein. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0080] The present disclosure also provides a molecular complex comprising any of the MST1R variant nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or a complement thereof, and any of the change-specific primers or change-specific probes described herein. In some embodiments, the MST1R variant nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) in the molecular complex, or a complement thereof, is single-stranded. In some embodiments, the MST1R variant nucleic acid molecule is any of the variant genomic nucleic acid molecules described herein. In some embodiments, the MST1R variant nucleic acid molecule is any of the variant mRNA molecules described herein. In some embodiments, the MST1R variant nucleic acid molecule is any of the variant cDNA molecules described herein. In some embodiments, the molecular complex comprises any of the MST1R variant nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or a complement thereof, and any of the change-specific primers described herein. In some embodiments, the molecular complex comprises any of the MST1R variant nucleic acid molecules (genomic nucleic acid molecules, mRNA molecules, or cDNA molecules) described herein, or a complement thereof, and any of the change-specific probes described herein. In some embodiments, the molecular complex comprises a non-human polymerase. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys or Arg651STOP. In some embodiments, the MST1 variant nucleic acid molecule is Arg703Cys. In some embodiments, the MST1 variant nucleic acid molecule is Arg651STOP. In some embodiments, the MST1R variant nucleic acid molecule is any of the MST1R variant nucleic acid molecules described herein.

[0081] All patent documents, websites, other publications, accession numbers, etc., cited above or below are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual item had been specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with different accession numbers at different times, the version associated with the accession number on the effective filing date of the present application is meant. The effective filing date means, where applicable, the earlier of the actual filing date or the filing date of the priority claim application for which the accession number is being referred to. Similarly, where different versions of a publication, website or the like have been published at different times, unless otherwise specified, the latest published version as of the effective filing date of the present application is meant. Any feature, step, element, embodiment, or aspect of the present disclosure can be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise specifically specified. The present disclosure has been described in some detail by way of examples and embodiments for purposes of clarity and understanding, but it will be apparent that certain changes and modifications can be made within the scope of the appended claims.

[0082] The following examples are provided to explain the embodiments in more detail. These are intended to illustrate, not to limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be merely exemplary and not to limit any of the claims. With respect to numbers (e.g., amounts, temperatures, etc.), efforts have been made to ensure accuracy, but some error and deviation may be considered. Unless otherwise specified, parts are by weight, temperature is in °C or ambient temperature, and pressure is at or near atmospheric pressure.

Example

[0083] Example 1: Collective Load Table 1 shows that the set of all rare predicted loss-of-function (pLOF) and predicted missense variants is significantly associated with increased odds of IBD in an exome-wide analysis of 4,319 IBD cases and 4,388 controls within the IBD cohort at the University of Kiel (odds ratio 2.99; p-value 1.62E-06). Table 1 also shows that the set of all rare predicted loss-of-function variants is associated with increased odds of IBD in an exome-wide analysis of 4,319 IBD cases and 4,388 controls within the IBD cohort at the University of Kiel (odds ratio 3.90; p-value 2.15E-04) (see also Figure 2).

[0084]

Table 1

[0085] Table 2 shows that the associations of the set of all predicted loss-of-function variants are replicated with odds ratios of 2.22 (p-value 0.018) and 3.21 (p-value 0.011), respectively, in the UK Biobank IBD cohort that includes 396 cases and 137,000 controls. Table 2 also shows that the associations of the set of all rare predicted loss-of-function and predicted missense variants and the set of all rare predicted loss-of-function variants are replicated with an odds ratio of 2.21 (p-value 0.048) in the UK Biobank IBD cohort that includes 494 cases and 30,000 controls.

[0086]

Table 2

[0087] The meta-analysis confirmed the association of this rare variant of this large effect between rare loss-of-function variants in MST1R for Crohn's disease (OR = 3.12) and PSC (OR = 5.59) (see Figure 10).

[0088] The association between individual MST1R variants and Crohn's disease is shown in Table 3.

[0089]

Table 3

[0090] Example 2: Mouse model of IBD (predictive example) Determine the effect of loss of MST1R signaling on the increased risk in the IBD mouse model. Generate MST1R KO mice and challenge them in a mouse model of IBD. Mice lacking MST1R are expected to be more prone to the mouse model of IBD. Also, generate MST1R transgenic mice with constitutive MST1R signaling. These mice are also expected to be protected in the mouse model of IBD.

[0091] In vitro experiments are also performed to determine whether the MST1 protein expressing rs3197999 binds to MST1R and signals through it. Generate a recombinant protein expressing MST1 missense rs3197999 and treat macrophage cell lines expressing normal MST1R with this protein and the wild-type protein. Determine whether the rs3197999 MST1 protein can activate MST1R signaling compared to the wild-type MST1 protein through measurement of signaling downstream of MST1R phospho-AKT / PI3K signaling. Also, generate macrophage cell lines expressing MST1R loss-of-function / missense variants and determine whether these mutant proteins can signal by measuring downstream phospho-AKT / PI3K signaling molecules in the presence of exogenous wild-type MST1 ligand.

[0092] Example 3: Novel MST1pLoF mutations are associated with IBD and PSC The common MST1 missense variant (rs3197999) is associated with increased odds of IBD and PSC; MST1 rs3197999 / p.Arg703Cys (allele frequency of 29%) is associated with decreased serum MST1 protein levels (Table 4).

[0093]

Table 4

[0094] A novel MST1 pLoF variant (3:49684379:G:A; c.1951C>T; p.Arg651* that generates a stop gain) associated with a greater impact on PSC and IBD disease risk was identified (Table 5 and Figure 3), confirming that LoF of MST1 increases disease risk. This effect is greater than that of MST1 missense variants.

[0095]

Table 5

[0096] Serum levels of MST1 were measured in MST1 variant carriers and controls. Serum was obtained from heterozygous and homozygous carriers of MST1 p.R703C, heterozygous and homozygous MST1 p.651*, and reference carriers (those not carrying either the MST1 p.R703C nor the MST1 p.651* variant). Circulating serum MST1 levels were measured using the Luminex assay. Serum levels of variant MST1 protein carriers were analyzed. The results show that both MST1 p.R703C missense carriers and novel p.651* pLoF variant carriers have significantly decreased levels of MST1 in serum compared to control reference carriers (Figure 9). The assay used is disclosed on the World Wide Web at "rndsystems.com / products / human-magnetic-luminex-assay_lxsahm".

[0097] Example 4: Gastrointestinal disorders result in changes in MST1 and MST1R expression in mice and human patients. The levels of MST1 and MST1R mRNA were examined in mice treated with DSS to induce colitis. Bulk RNAseq assays revealed increased expression of both MST1 and MST1R in a mouse model of colitis (Figure 4).

[0098] MST1 serum levels were evaluated in human IBD patients compared to healthy controls (Figure 5). Serum was obtained from IBD patients and healthy controls (who did not carry the MST1 p.R703C, MST1 p.651*, or MST1R pLOF variants), and MST1 was measured using a Luminex cytokine assay (as described above). Analysis of these samples showed an increase in MST1 serum concentration among IBD patients compared to healthy controls. MST1 and MST1R were also increased in patients with Crohn's disease, ulcerative colitis, and functional gastrointestinal disorders (FGID) (Figure 6). Single-cell RNA sequencing analysis was performed on intestinal biopsies from patients with either Crohn's disease (CD), ulcerative colitis (UC), or FGID as a control. Both MST1 and MST1R showed higher expression in small intestinal epithelial cells of human CD patients than in intestinal mucosal lamina propria cells of human FGID patients (Figure 7).

[0099] In contrast to the increased wild-type MST1 / MST1R levels observed in IBD patients, IBD patients carrying variant MST1 or MST1R showed decreased serum MST1 levels (Figure 9). There was no difference between IBD and control levels among variant carriers.

[0100] Example 5: Expression of variant MST1R constructs To determine whether the ATG codon encoding methionine at position 464 of the MST1R protein can be used as the translation start codon, the expression of the MST1R cDNA construct carrying the G116fs mutation was examined. Western blot analysis of HEK cell lines transduced with cDNA encoding either full-length, wild-type MST1R or MST1R G116fs showed that the introduction of a frameshift in the MST1R cDNA resulted in the translation of a short truncated protein, indicating that the downstream in-frame ATG was not activated (Figure 11).

[0101] To analyze the properties of the truncated MST1R pLoF protein, HEK cells were transfected with constructs encoding either WT MST1R cDNA or cDNA encoding MST1R carrying the frameshift G116fs mutation. Western blot analysis of lysates generated from these cell lines showed that WT MST1R was strongly expressed and activated its downstream signaling targets including phosphorylated ERK1 / 2 (pERK1 / 2), phosphorylated AKT (pAKT), and phosphorylated MST1R (pMST1R), whereas the expression of the mutant MST1R was not detected and no activation of downstream targets was observed (Figure 12), demonstrating that the MST1Rfs mutant is a true loss-of-function mutant.

[0102] Example 6: Expression of Variant MST1 Constructs The results of Example 1 showed that the collection of rare pLoFs / missense variants in MST1R is associated with an increased risk of IBD, suggesting that loss of MST1R increases the risk of IBD. Increased MST1 protein levels and / or increased MST1 / MST1R signaling may be protective and / or promote tissue repair in IBD and PSC.

[0103] To measure the potential of MST1 expression, several constructs encoding WT and variant MST1 cDNAs (Figure 13) were transfected into HEK cells. Western blot analysis showed that all of the variant MST1 constructs were expressed and could be secreted (Figure 14).

[0104] Using hydrodynamic delivery (HDD), WT MST1 cDNA packaged in the expression construct was delivered to mice, resulting in overexpression of WT MST1. The fact that the mice injected with the MST1 HDD construct survived the course of the experiment indicates that MST1 overexpression is not lethal (data not shown). Consistent expression of the control HDD construct was observed in the livers of three mice, and consistent expression of the mMst1 construct was observed in the livers of three mMst1-HDD mice, indicating that these constructs were expressed and that the expression was consistent in each group (Figure 8). Also, MST1 overexpression did not seem to affect mouse body weight (Figure 15). As shown in a Western blot for the protein tag included as part of the construct (Figure 16), MST1 plasma levels were below the detection level. The tag was likely cleaved and not detected. The vector was detectable when expressed in the CHO cell line.

[0105] In addition to what is described herein, various modifications of the described subject matter will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, GenBank accession numbers, and the like) is hereby incorporated by reference in its entirety for all purposes. Exemplary embodiments 1. A method of treating a patient having inflammatory bowel disease (IBD), the method comprising administering to the patient an agonist of the macrophage stimulating 1 (MST1) / macrophage stimulating 1 receptor (MST1R) pathway. 2. A method of treating a patient having primary sclerosing cholangitis (PSC), the method comprising administering to the patient an agonist of the macrophage stimulating 1 (MST1) / macrophage stimulating 1 receptor (MST1R) pathway. 3. The method according to embodiment 1 or embodiment 2, wherein the agonist of the MST1 / MST1R pathway is an MST1 agonist or an MST1R agonist. 4. The method according to embodiment 3, wherein the MST1 agonist and / or the MST1R agonist comprises a protein, a nucleic acid molecule, or a small molecule. 5. The method according to embodiment 4, wherein the protein is recombinant MST1. 6. The method according to embodiment 4, wherein the protein is an antibody against MST1 or MST1R. 7. The method according to embodiment 3, wherein the MST1 agonist is kereberin, recombinant tumor necrosis factor receptor-associated factor 2 (TRAF2), or curcumin. 8. The method according to embodiment 3, wherein the MST1 agonist is a protease selected from hepatocyte growth factor activator (HGFA), matriptase, hepsin, TMPRSS11D (human airway trypsin-like protease; HAT), coagulation factor XIIa, coagulation factor Xia, and kallikrein. 9. The method according to embodiment 3, wherein the MST1R agonist is recombinant macrophage scavenger receptor 1 (MSR1), recombinant hepatocyte growth factor-like protein (HGFL), or recombinant androgen receptor (AR). 10. The method according to embodiment 3, wherein the MST1R agonist is an antibody selected from mAb Zt / g4, mAb Zt / c1, mAb Zt / f2, mAb Zt / 64, mAb 3F12, mAb B9, and mAb 1G4. 11. The method according to any one of embodiments 1 to 10, further comprising detecting the presence or absence of an MST1 and / or MST1R variant nucleic acid molecule or variant polypeptide associated with an increased risk of developing IBD and / or PSC in a biological sample from the patient. 12. The method according to embodiment 11, wherein the MST1 variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC encodes Arg703Cys or Arg651STOP. 13. The method according to embodiment 11, wherein the MST1R variant nucleic acid molecule associated with an increased risk of developing IBD and / or PSC is 3:49903264:CG:C, 3:49903084:C:T, 3:49890026:G:T, 3:49902417:A:G, 3:49902560:A:T, and / or 3:49903387:G:T, or an mRNA molecule generated therefrom, or a cDNA molecule generated from the mRNA molecule. 14. Detecting the presence or absence of an MST1 and / or MST1R variant nucleic acid molecule or variant polypeptide associated with an increased risk of developing IBD and / or PSC comprises determining whether the patient has an MST1 and / or MST1R variant genomic nucleic acid molecule associated with an increased risk of developing IBD and / or PSC, an MST1 and / or MST1R variant mRNA molecule associated with an increased risk of developing IBD and / or PSC, an MST1 and / or MST1R variant cDNA molecule generated from the mRNA molecule, and / or an MST1 and / or MST1R variant polypeptide associated with an increased risk of developing IBD and / or PSC, obtaining or having obtained a biological sample from the patient, and performing or having performed an assay on the biological sample to determine whether the patient has an MST1 and / or MST1R variant nucleic acid molecule or variant polypeptide associated with an increased risk of developing IBD and / or PSC. The method according to any one of Embodiments 11 to 13, including determining by 15. The method according to Embodiment 14, further comprising determining the population burden of a patient having an MST1 and / or MST1R variant genomic nucleic acid molecule associated with an increased risk of developing IBD and / or PSC, an MST1 and / or MST1R variant mRNA molecule associated with an increased risk of developing IBD and / or PSC, an MST1 and / or MST1R variant cDNA molecule generated from said mRNA molecule, and / or an MST1 and / or MST1R variant polypeptide associated with an increased risk of developing IBD and / or PSC. 16. The method according to Embodiment 14 or Embodiment 15, wherein said assay is a genotyping assay for nucleic acid molecules. 17. The method according to Embodiment 14 or Embodiment 15, wherein said assay is an immunoassay for polypeptides. 18. The method according to any one of Embodiments 11 to 17, wherein said step of detecting, determining, or assay is performed in vitro. 19. The method according to any one of Embodiments 11 to 18, wherein said step of detecting, determining, or assay comprises sequencing at least a part of the nucleotide sequence of the MST1 and / or MST1R nucleic acid molecule in said biological sample, and the part to be sequenced includes a position corresponding to a predicted loss-of-function variant position, and when a variant nucleotide at the predicted loss-of-function variant position is detected, the MST1 and / or MST1R nucleic acid molecule in said biological sample is a predicted loss-of-function variant nucleic acid molecule of MST1 and / or MST1R. 20. The step of detecting, determining, or assay is a) contacting said biological sample with a primer that hybridizes to a part of the nucleotide sequence of said MST1 and / or MST1R nucleic acid molecule proximate to a predicted loss-of-function variant position b) extending the primer through at least the predicted loss-of-function variant position, and c) determining whether the extension product of the primer contains a variant nucleotide at the predicted loss-of-function variant position The method according to embodiment 19, comprising: 21. The method according to embodiment 19 or embodiment 20, wherein the detecting step, the determining step, or the assay comprises sequencing the entire nucleic acid molecule. 22. The method according to any one of embodiments 11-18, wherein the detecting step, the determining step, or the assay comprises sequencing at least a portion of the nucleotide sequence of the MST1 and / or MST1R nucleic acid molecule in the biological sample, and the portion to be sequenced comprises a position corresponding to a missense variant position, and when a variant nucleotide at the missense variant position is detected, the MST1 and / or MST1R nucleic acid molecule in the biological sample is an MST1 and / or MST1R missense variant nucleic acid molecule. 23. The detecting step, the determining step, or the assay comprises: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the MST1 and / or MST1R nucleic acid molecule adjacent to the missense variant position; b) extending the primer through at least the missense variant position; and c) determining whether the extension product of the primer contains a variant nucleotide at the missense variant position. The method according to embodiment 22, comprising: 24. The method according to embodiment 22 or embodiment 23, wherein the detecting step, the determining step, or the assay comprises sequencing the entire nucleic acid molecule. 25. The detecting step, the determining step, or the assay comprises: a) Amplifying at least a part of the MST1 and / or MST1R nucleic acid molecule, wherein the part includes the predicted loss-of-function variant position, said amplifying; b) Labeling the amplified nucleic acid molecule with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the predicted loss-of-function variant position, said contacting, and d) Detecting the detectable label The method according to any one of Embodiments 11 to 18, comprising: 26. The method according to Embodiment 25, wherein the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA before the amplifying step. 27. The detecting step, determining step, or assay is Contacting the nucleic acid molecule in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the predicted loss-of-function variant position, said contacting, and Detecting the detectable label The method according to Embodiment 25 or Embodiment 26, comprising: 28. The detecting step, determining step, or assay is a) Amplifying at least a part of the MST1 and / or MST1R nucleic acid molecule, wherein the part includes the missense variant position, said amplifying; b) Labeling the amplified nucleic acid molecule with a detectable label; c) Contacting the labeled nucleic acid molecule with a support comprising a change-specific probe, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to the missense variant position, said contacting, and d) Detecting the detectable label The method according to any one of Embodiments 11 to 18, comprising 29. The method according to Embodiment 28, wherein the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA before the step of amplification. 30. The step of detecting, determining, or assay is contacting the nucleic acid molecule in the biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a missense variant position, the contacting, and detecting the detectable label The method according to Embodiment 28 or Embodiment 29, comprising

Claims

[Claim 1] A method of treating a patient having inflammatory bowel disease (IBD), comprising administering to the patient an agonist of the macrophage stimulating 1 (MST1) / macrophage stimulating 1 receptor (MST1R) pathway.

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