Treatment of liver diseases with cAMP-responsive element-binding protein 3-like 3 (CREB3L3) inhibitors

Abstract

The present disclosure provides methods of treating a subject having liver disease with a cAMP-responsive element binding protein 3-like 3 (CREB3L3) inhibitor, and methods of identifying subjects at high risk for developing liver disease.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as an XML file of 242 kilobytes in size under the title 381203558SEQ, created on August 23, 2022. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to the treatment of subjects with liver disease with cAMP-responsive element binding protein 3-like 3 (CREB3L3) inhibitors, and to methods of identifying subjects at high risk for developing liver disease. [Background technology]

[0003] Chronic liver disease and cirrhosis are the leading causes of morbidity and mortality in the United States, accounting for 38,170 deaths in 2014 (1.5% of total deaths) (Non-Patent Document 1). In the United States, the most common causes of cirrhosis are alcoholic liver disease, chronic hepatitis C, and non-alcoholic fatty liver disease (NAFLD), which accounted for approximately 80% of patients waiting for liver transplants between 2004 and 2013 (Non-Patent Document 2). The estimated prevalence of NAFLD in the United States is 19-46 percent (Non-Patent Document 3; Non-Patent Document 4 and Non-Patent Document 5), and has been increasing over time, likely in conjunction with rising obesity rates, one of the main risk factors for NAFLD (Non-Patent Document 6) (Non-Patent Document 7). Although there have been great advances in the treatment of hepatitis C, there are currently no evidence-based treatments for alcoholic or non-alcoholic liver disease or cirrhosis. Identifying naturally occurring genetic variants that protect against liver damage and liver disease outcomes may be a route to identifying novel therapeutic targets for liver disease (Non-Patent Document 8).

[0004] CREB3L3 is a member of the basic leucine zipper family and the AMP-dependent transcription factor family. CREB3L3 localizes to the endoplasmic reticulum and acts in response to cAMP stimulation during endoplasmic reticulum stress by activating the transcription of unfolded protein response target genes via box-B elements. In vitro, CREB3L3 binds to cyclic AMP response elements (CRE) and box-B elements and is associated with acute inflammatory responses, hepatocellular carcinoma, triglyceride metabolism, and hepcidin expression. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kochanek et al.,Nat'l. Vital Stat. Rep.,2016,65,1-122 [Non-Patent Document 2] Wong et al.,Gastroenterology,2015,148,547-555 [Non-Patent Document 3] Browning et al.,Hepatology,2004,40,1387-1395 [Non-Patent Document 4] Lazo et al.,Am. J. Epidemiol.,2013,178,38-45 [Non-Patent Document 5] Williams et al.,Gastroenterology,2011,140,124-131 [Non-Patent Document 6] Cohen et al.,Science,2011,332,1519-1523 [Non-Patent Document 7] Younossi et al.,Clin.Gastroenterol.Hepatol.,2011,9,524-530 [Non-Patent Document 8] Abul-Husn et al.N.Engl. J.Med.,2018,378,1096-106 Summary of the Invention

[0006] The present disclosure provides a method of treating a subject having liver disease, the method comprising administering to the subject a CREB3L3 inhibitor. The disclosure also provides a method of treating a subject having parenchymal liver disease, the method comprising administering to the subject a CREB3L3 inhibitor.

[0007] The present disclosure also provides a method of treating a subject having liver fibrosis, the method comprising administering to the subject a CREB3L3 inhibitor. The present disclosure also provides a method of treating a subject with cirrhosis, the method comprising administering to the subject a CREB3L3 inhibitor.

[0008] The present disclosure also provides a method of treating a subject having non-alcoholic fatty liver disease (NAFLD), the method comprising administering to the subject a CREB3L3 inhibitor. The disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits liver disease, wherein the subject has liver disease, the method comprising: determining whether the subject has a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in CREB3L3 by obtaining or obtaining a biological sample from the subject; performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in CREB3L3; and administering to the subject a CREB3L3 variant that is a genotype that includes a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in CREB3L3; administering or continuing to administer a therapeutic agent that treats or inhibits liver disease at a standard dosage, and administering a CREB3L3 inhibitor to the subject; administering or continuing to administer a therapeutic agent that treats or inhibits liver disease at the same or a lower standard dosage, to a subject who is heterozygous for a CREB3L3 mutant nucleic acid molecule, and administering a CREB3L3 inhibitor to the subject; wherein the presence of a genotype having a CREB3L3 mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3 indicates that the subject is at low risk of developing liver disease.

[0009] The present disclosure also provides a method for identifying a subject at high risk of developing liver disease, the method comprising determining or having determined the presence or absence of a CREB3L3 mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted in CREB3L3 in a biological sample obtained from the subject; wherein if the subject is a CREB3L3 norm, the subject has a high risk of developing liver disease; and if the subject is heterozygous or homozygous for a CREB3L3 mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted in CREB3L3, the subject has a low risk of developing liver disease.

[0010] The present disclosure also provides a therapeutic agent for treating or inhibiting liver disease, comprising a genomic nucleic acid molecule encoding a predicted loss-of-function polypeptide of CREB3L3 having a nucleotide sequence including an adenine at position corresponding to position 6,120 set forth in SEQ ID NO:2, or a complement thereof; an adenine at position corresponding to position 661 set forth in SEQ ID NO:17, or a complement thereof; an adenine at position corresponding to position 649 set forth in SEQ ID NO:18, or a complement thereof; an adenine at position corresponding to position 624 set forth in SEQ ID NO:19, or a complement thereof; an adenine at a position corresponding to position 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:26, or its complement. an mRNA molecule encoding a predicted loss-of-function polypeptide of CREB3L3 having a nucleotide sequence comprising an adenine, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:28, or its complement; or an adenine at a position corresponding to position 661 set forth in SEQ ID NO:45, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:46, or its complement; an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:48; an adenine, or its complement, at a position corresponding to position 658 in SEQ ID NO:49; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:50; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:51; an adenine, or its complement, at a position corresponding to position 663 in SEQ ID NO:52; an adenine, or its complement, at a position corresponding to position 660 in SEQ ID NO:53;The present invention provides a therapeutic agent for use in treating liver disease in a subject identified as having a cDNA molecule encoding a predicted loss-of-function polypeptide of CREB3L3, or a complement thereof, having a nucleotide sequence including an adenine at a position corresponding to 649 as set forth in SEQ ID NO:54, or a complement thereof; an adenine at a position corresponding to 624 as set forth in SEQ ID NO:55, or a complement thereof; or an adenine at a position corresponding to 691 as set forth in SEQ ID NO:56, or a complement thereof;

[0011] The present disclosure also provides a CREB3L3 inhibitor, comprising: a) a CREB3L3 genomic nucleic acid molecule, a CREB3L3 mRNA molecule, or a CREB3L3 adenine at a position corresponding to position 6,120 as set forth in SEQ ID NO:2, or its complement; ii) an adenine at a position corresponding to position 661 as set forth in SEQ ID NO:17, or its complement; an adenine at a position corresponding to position 649 as set forth in SEQ ID NO:18, or its complement; an adenine at a position corresponding to position 624 as set forth in SEQ ID NO:19, or its complement; an adenine at a position corresponding to position 624 as set forth in SEQ ID NO:20, or its complement; an adenine at a position corresponding to position 658 as set forth in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 as set forth in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 as set forth in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 as set forth in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 as set forth in SEQ ID NO:25, or its complement; an mRNA molecule encoding a predicted loss-of-function polypeptide of CREB3L3 having a nucleotide sequence comprising an adenine at a position corresponding to position 649 of SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 of SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 of SEQ ID NO:28, or its complement; or iii) an adenine at a position corresponding to position 661 of SEQ ID NO:45, or its complement. an adenine at a position corresponding to position 649 in SEQ ID NO:46, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:47, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:48, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:49, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:50, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:51, or its complement;The present invention provides a CREB3L3 inhibitor for use in treating liver disease in a subject who is heterozygous for a cDNA molecule encoding a CREB3L3 predicted loss-of-function polypeptide having a nucleotide sequence comprising: an adenine at a position corresponding to 663 as set forth in SEQ ID NO:52, or its complement; an adenine at a position corresponding to 660 as set forth in SEQ ID NO:53, or its complement; an adenine at a position corresponding to 649 as set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to 624 as set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to 691 as set forth in SEQ ID NO:56, or its complement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Various terms relating to the aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other terms that are specifically defined are to be interpreted in a manner consistent with the definitions set forth herein.

[0013] Unless expressly stated otherwise, any method or embodiment set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically states in the claims or detailed description that the steps are to be limited to a particular order, it is in no way intended to dictate order in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or work flow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0014] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that a cited numerical value is approximate, and small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0015] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0016] As used herein, with respect to a nucleic acid molecule or polypeptide, the term "isolated" means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., away from blood and / or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form, i.e., more than 95% pure or more than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternatively phosphorylated or derivatized forms.

[0017] As used herein, the terms "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 multistranded. A strand of a nucleic acid also refers to its complement.

[0018] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates (e.g., apes and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0019] Partial loss of function of the CREB3L3 gene has been identified according to the present disclosure, which is associated with a reduced risk of developing liver disease in humans. For example, it has been observed that a genetic mutation that changes the guanine to adenine at position 6,120 of the CREB3L3 reference genomic nucleic acid molecule (see SEQ ID NO: 1) indicates that subjects with such a mutation may have a lower risk of developing liver disease. It is believed that CREB3L3 gene or protein variants have no known association with human liver disease. In summary, the genetic analysis described herein surprisingly shows that the CREB3L3 gene, and in particular, variants of the CREB3L3 gene, are associated with a reduced risk of developing liver disease. Furthermore, the identification of the association of additional variants with the genetic burden mask according to the present disclosure indicates that CREB3L3 itself (rather than linkage disequilibrium with variants of another gene) is responsible for the protective effect in liver disease. Thus, subjects with CREB3L3 criteria at high risk of developing liver disease, such as parenchymal liver disease, liver fibrosis, cirrhosis, or NAFLD, can be treated to prevent liver disease, reduce symptoms, and / or inhibit the onset of symptoms. Thus, the present disclosure provides a method that utilizes the identification of such variants in a subject to identify or stratify the risk in such a subject of developing liver disease, such as parenchymal liver disease, liver fibrosis, cirrhosis, or NAFLD, or to diagnose a subject as having a high risk of developing liver disease, such as parenchymal liver disease, liver fibrosis, cirrhosis, or NAFLD, so that subjects at risk or with active disease can be treated accordingly.

[0020] For the purpose of this disclosure, any particular subject can be classified as having one of three CREB3L3 genotypes: i) CREB3L3 standard; ii) heterozygous for CREB3L3 variant nucleic acid molecule encoding loss-of-function polypeptide predicted by CREB3L3; or iii) homozygous for CREB3L3 variant nucleic acid molecule encoding loss-of-function polypeptide predicted by CREB3L3.If the subject does not have a copy of CREB3L3 variant nucleic acid molecule encoding loss-of-function polypeptide predicted by CREB3L3, the subject is CREB3L3 standard.If the subject has a single copy of CREB3L3 variant nucleic acid molecule encoding loss-of-function polypeptide predicted by CREB3L3, the subject is heterozygous for CREB3L3 variant nucleic acid molecule encoding loss-of-function polypeptide predicted by CREB3L3. As used herein, a CREB3L3 variant nucleic acid molecule is any CREB3L3 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) that encodes a CREB3L3 polypeptide with partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. A subject that has a CREB3L3 variant nucleic acid molecule that encodes a predicted CREB3L3 loss of function polypeptide with partial loss of function (or predicted partial loss of function) is hypomorphic for CREB3L3. A CREB3L3 variant nucleic acid molecule that encodes a predicted CREB3L3 loss of function polypeptide can be any nucleic acid molecule that encodes CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, Asp182Asn-D, or Asp181Asn. In some embodiments, the CREB3L3 mutant nucleic acid molecule encodes CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, or Asp182Asn-D. If a subject has two copies of a CREB3L3 mutant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3, the subject is homozygous for the CREB3L3 mutant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3.

[0021] For subjects who are genotyped or determined to be CREB3L3 standard, such subjects are at high risk of developing liver disease, such as parenchymal liver disease, liver fibrosis, liver cirrhosis, or NAFLD.For subjects who are genotyped or determined to be CREB3L3 standard or heterozygous for a CREB3L3 variant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3, such subjects can be treated with a CREB3L3 inhibitor.

[0022] In any of the embodiments described throughout this disclosure, the CREB3L3 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CREB3L3 can be any CREB3L3 nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding a CREB3L3 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. For example, the CREB3L3 mutant nucleic acid molecule can be any nucleic acid molecule encoding CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, Asp182Asn-D, or Asp181Asn. In some embodiments, the CREB3L3 mutant nucleic acid molecule encodes CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, or Asp182Asn-D.

[0023] In any of the embodiments described throughout this disclosure, the predicted loss-of-function polypeptide for CREB3L3 can be any CREB3L3 polypeptide with 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 throughout this disclosure, the predicted loss-of-function polypeptide for CREB3L3 can be any of the CREB3L3 polypeptides described herein, including, for example, CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, Asp182Asn-D, or Asp181Asn. In some embodiments, the predicted loss-of-function polypeptide for CREB3L3 is CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, or Asp182Asn-D.

[0024] In any of the embodiments described throughout this disclosure, the liver disease is a fatty liver disease (e.g., alcoholic fatty liver disease (AFLD), NAFLD or non-alcoholic steatohepatitis (NASH)), cirrhosis, liver fibrosis, elevated liver enzymes (e.g., alanine transaminase (ALT) or aspartate transaminase (AST)), simple fatty liver, steatohepatitis, parenchymal liver disease, viral hepatitis, or hepatocellular carcinoma, or any of the complications of such conditions (including, but not limited to, cardiac or metabolic disease associated with NASH or NAFLD, portal hypertension or thrombosis, esophageal or gastric varices or bleeding therefrom, and other co-morbidities associated with liver disease). In some embodiments, the liver disease is fatty liver disease. In some embodiments, the liver disease is AFLD. In some embodiments, the liver disease is NAFLD. In some embodiments, the liver disease is NASH. In some embodiments, the liver disease is cirrhosis. In some embodiments, the liver disease is liver fibrosis. In some embodiments, the liver disease is elevated liver enzymes. In some embodiments, the liver disease is elevated ALT. In some embodiments, the liver disease is elevated AST. In some embodiments, the liver disease is simple fatty liver. In some embodiments, the liver disease is steatohepatitis. In some embodiments, the liver disease is parenchymal liver disease. In some embodiments, the liver disease is viral hepatitis. In some embodiments, the liver disease is hepatocellular carcinoma. In some embodiments, the liver disease is liver damage quantified by liver biomarkers (e.g., liver transaminases), changes in liver biomarkers, or liver imaging.

[0025] Symptoms of liver disease include, but are not limited to, liver enlargement, fatigue, pain in the upper right abdomen, abdominal distension (ascites), dilation of blood vessels just below the surface of the skin, male breast enlargement, spleen enlargement, palmar erythema, yellowing of the skin and eyes (jaundice), pruritus, dark urine, pale stool, nausea or vomiting, loss of appetite, and tendency to bruise easily.Testing for liver disease can involve blood tests, liver imaging, and liver biopsy.If a subject has at least one known risk factor (e.g., genetic factor such as pathogenic mutation), the individual is at increased risk of developing liver disease, and the individual with the risk factor is ranked as a person who is at a statistically significant higher risk of developing the disease than the individual without the risk factor. Risk factors for liver disease include, for example, excessive alcohol consumption, obesity, high cholesterol, high levels of blood triglycerides, polycystic ovary syndrome, sleep apnea, type 2 diabetes, an underactive thyroid gland (hypothyroidism), an underactive pituitary gland (hypopituitarism), and metabolic syndrome (including elevated blood lipids).

[0026] The present disclosure provides a method of treating a subject having liver disease, the method comprising administering to the subject a CREB3L3 inhibitor. The disclosure also provides a method of treating a subject having parenchymal liver disease, the method comprising administering to the subject a CREB3L3 inhibitor.

[0027] The present disclosure also provides a method of treating a subject having liver fibrosis, the method comprising administering to the subject a CREB3L3 inhibitor. The present disclosure also provides a method of treating a subject with cirrhosis, the method comprising administering to the subject a CREB3L3 inhibitor.

[0028] The present disclosure also provides a method of treating a subject having NAFLD, the method comprising administering to the subject a CREB3L3 inhibitor. In some embodiments, the CREB3L3 inhibitor comprises an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule, a small interfering RNA (siRNA) molecule, or a short hairpin RNA (shRNA) molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an shRNA molecule. Such inhibitory nucleic acid molecules can be designed to target any region of a CREB3L3 nucleic acid molecule, such as an mRNA molecule. In some embodiments, the inhibitory nucleic acid molecule hybridizes with a sequence within a CREB3L3 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of a CREB3L3 polypeptide in a cell of a subject. In some embodiments, the CREB3L3 inhibitor comprises an antisense molecule that hybridizes with a CREB3L3 genomic nucleic acid molecule or an mRNA molecule, and reduces the expression of a CREB3L3 polypeptide in a cell of a subject. In some embodiments, the CREB3L3 inhibitor comprises an siRNA that hybridizes with a CREB3L3 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of a CREB3L3 polypeptide in a subject's cell. In some embodiments, the CREB3L3 inhibitor comprises an shRNA that hybridizes with a CREB3L3 genomic nucleic acid molecule or an mRNA molecule and reduces the expression of a CREB3L3 polypeptide in a subject's cell.

[0029] In some embodiments, the CREB3L3 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) in the CREB3L3 genomic nucleic acid molecule or in a DNA binding protein that binds to the recognition sequence. The recognition sequence can be located in the coding region of the CREB3L3 gene or in a regulatory region that affects the expression of the gene. The recognition sequence of the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence can include or be adjacent to the start codon of the CREB3L3 gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each of which targets a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence containing or adjacent to a start codon, and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and cleavage by these nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences.Any nuclease agent that induces a nick or double-strand break at the desired recognition sequence can be used in the methods and compositions disclosed herein.Any DNA binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0030] Suitable nuclease agents and DNA binding proteins for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, but includes, for example, recognition sequences that are about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNA.

[0031] In some embodiments, the CRISPR / Cas system can be used to modify the CREB3L3 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can employ the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the CREB3L3 nucleic acid molecule.

[0032] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins can also contain nuclease domains (e.g., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (e.g., FnCpf1). Cas proteins can have full cleavage activity to create double-stranded breaks in CREB3L3 genomic nucleic acid molecules, or can be nickases to create single-stranded breaks in CREB3L3 genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cas proteins include, but are not limited to, Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or variants thereof. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Cas proteins can be provided in any form. For example, the Cas protein can be provided in the form of a protein, e.g., a Cas protein complexed with a gRNA.Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule, e.g., RNA or DNA, encoding the Cas protein.

[0033] In some embodiments, targeted genetic modification of a CREB3L3 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in a CREB3L3 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within a region of SEQ ID NO:1. The gRNA recognition sequence can also include or be adjacent to a position corresponding to position 6,120 as set forth in SEQ ID NO:1. For example, the gRNA recognition sequence can be located about 1000, about 500, about 400, about 300, about 200, about 100, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides away from a position corresponding to position 6,120 as set forth in SEQ ID NO:1. The gRNA recognition sequence can include or be adjacent to a start codon of a CREB3L3 genomic nucleic acid molecule or a stop codon of a CREB3L3 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from the start codon or the stop codon.

[0034] The gRNA recognition sequence in the target genomic locus in the CREB3L3 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. A canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. The gRNA can transport Cas9 anywhere in the genome for gene editing, but cannot edit at sites other than the site where Cas9 recognizes the PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is about 2 to about 6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 3' end. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10 base pairs, about 2 to about 5 base pairs, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (e.g., when using Cas9 from S. pyogenes or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand is 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0035] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific position in CREB3L3 genomic nucleic acid molecule. An exemplary gRNA is an effective gRNA for inducing Cas enzyme to bind to or cleave CREB3L3 genomic nucleic acid molecule, wherein the gRNA comprises a DNA targeting segment that hybridizes with a gRNA recognition sequence in CREB3L3 genomic nucleic acid molecule that includes or is adjacent to the position corresponding to position 6,120 in SEQ ID NO:1. For example, the gRNA can be selected to hybridize with a gRNA recognition sequence that is located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from the position corresponding to position 6,120 in SEQ ID NO:1. Other exemplary gRNAs comprise a DNA targeting segment that hybridizes with a gRNA recognition sequence present in the CREB3L3 genomic nucleic acid molecule that includes or is adjacent to the start codon or stop codon.For example, gRNAs can be selected to hybridize with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from the start codon, or with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from the stop codon. A suitable gRNA can comprise about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can comprise 20 nucleotides.

[0036] Examples of suitable gRNA recognition sequences located within the CREB3L3 reference gene are set forth in Table 1 as SEQ ID NOs: 69-88. Table 1: Guide RNA recognition sequences near CREB3L3 mutations

[0037] [Table 1]

[0038] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target CREB3L3 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target CREB3L3 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including a gRNA that hybridizes with a gRNA recognition sequence and complexes with a Cas protein) can result in the cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 or more base pairs) the nucleic acid sequence present in the CREB3L3 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0039] Such a method can produce a CREB3L3 genomic nucleic acid molecule in which, for example, a region of SEQ ID NO:1 is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the CREB3L3 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0040] In some embodiments, the CREB3L3 inhibitor comprises a small molecule. In some embodiments, the treatment method further comprises detecting the presence or absence of a CREB3L3 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CREB3L3 in a biological sample obtained from a subject.As used throughout this disclosure, a "CREB3L3 mutant nucleic acid molecule" is any CREB3L3 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a CREB3L3 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0041] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits liver disease. In some embodiments, the subject has liver disease. In some embodiments, the method includes determining whether the subject has a CREB3L3 mutant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3 by obtaining or obtaining a biological sample from the subject and performing or performing sequence analysis on the biological sample to determine whether the subject has a genotype that includes a CREB3L3 mutant nucleic acid molecule. If the subject is CREB3L3 standard, the therapeutic agent that treats or inhibits liver disease is administered or continues to be administered to the subject at a standard dosage, and a CREB3L3 inhibitor is administered to the subject. If the subject is heterozygous for the CREB3L3 mutant nucleic acid molecule, the therapeutic agent that treats or inhibits liver disease is administered or continues to be administered to the subject at a standard dosage or less, and a CREB3L3 inhibitor is administered to the subject. The presence of the genotype having the CREB3L3 variant nucleic acid molecule that encodes the loss-of-function polypeptide predicted by CREB3L3 indicates that the subject has a low risk of developing liver disease.In some embodiments, the subject is CREB3L3 standard.In some embodiments, the subject is heterozygous for the CREB3L3 variant nucleic acid molecule that encodes the loss-of-function polypeptide predicted by CREB3L3.

[0042] For subjects who have been genotyped or determined to be heterozygous for a CREB3L3 variant nucleic acid molecule that encodes a CREB3L3-based or CREB3L3-predicted loss-of-function polypeptide, such subjects can be treated with a CREB3L3 inhibitor as described herein.

[0043] Detecting the presence or absence of a CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in a biological sample from a subject and / or determining whether a subject has a CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in CREB3L3 can be performed by any of the methods described herein. In some embodiments, these methods can be 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 CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in CREB3L3 can be present in a cell obtained from the subject.

[0044] In some embodiments, if the subject is CREB3L3 normative, the subject is also administered a standard dose of a therapeutic agent for treating or inhibiting liver disease.In some embodiments, if the subject is heterozygous for a CREB3L3 variant nucleic acid molecule that encodes a predicted loss-of-function polypeptide in CREB3L3, the subject is also administered a standard dose or a lower dose of a therapeutic agent for treating or inhibiting liver disease.

[0045] In some embodiments, the method of treatment further comprises detecting the presence or absence of a loss-of-function polypeptide predicted by CREB3L3 in the subject's biological sample. In some embodiments, if the subject does not have a loss-of-function polypeptide predicted by CREB3L3, the subject is also administered a standard dose of a therapeutic agent for treating or inhibiting liver disease. In some embodiments, if the subject has a loss-of-function polypeptide predicted by CREB3L3, the subject is also administered a standard dose of a therapeutic agent for treating or inhibiting liver disease.

[0046] The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits liver disease. In some embodiments, the subject has liver disease. In some embodiments, the method includes determining whether the subject has a CREB3L3 predicted loss-of-function polypeptide by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a CREB3L3 predicted loss-of-function polypeptide. If the subject does not have a CREB3L3 predicted loss-of-function polypeptide, the therapeutic agent that treats or inhibits liver disease is administered or continues to be administered to the subject at a standard dose, and a CREB3L3 inhibitor is administered to the subject. If the subject has a CREB3L3 predicted loss-of-function polypeptide, the therapeutic agent that treats or inhibits liver disease is administered or continues to be administered to the subject at the same or lower dose than the standard dose, and a CREB3L3 inhibitor is administered to the subject. The presence of a CREB3L3 predicted loss-of-function polypeptide indicates that the subject is at a low risk of developing liver disease. In some embodiments, the subject has a CREB3L3 predicted loss-of-function polypeptide. In some embodiments, the subject does not have a CREB3L3 predicted loss-of-function polypeptide.

[0047] Detecting the presence or absence of a CREB3L3 predicted loss-of-function polypeptide in a biological sample from a subject and / or determining whether a subject has a CREB3L3 predicted loss-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be 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 CREB3L3 predicted loss-of-function polypeptide can be present in a cell obtained from the subject.

[0048] Examples of therapeutic agents that treat or inhibit liver disease include, but are not limited to, disulfiram, naltrexone, acamprosate, prednisone, azathioprine, penicillamine, trientine, deferoxamine, ciprofloxacin, norfloxacin, ceftriaxone, ofloxacin, amoxicillin-clavulanate, phytonadione, bumetanide, furosemide, hydrochlorothiazide, chlorothiazide, amiloride, triamterene, spironolactone, octreotide, atenolol, metoprolol, nadolol, propranolol, timolol, and carvedilol, or any combination thereof.

[0049] Additional examples of liver disease therapeutics (e.g., for use in treating chronic hepatitis C) include, but are not limited to, ribavirin, paritaprevir, OLYSIO® (simeprevir), grazoprevir, ledipasvir, ombitasvir, elbasvir, DAKLINZA® (daclatasvir), dasabuvir, ritonavir, sovosbuvir, velpatasvir, voxilaprevir, glecaprevir, pibrentasvir, peginterferon alfa-2a, peginterferon alfa-2b, and interferon alfa-2b.

[0050] Additional examples of liver disease therapeutic agents (e.g., for use in NFLD) include, but are not limited to, weight loss inducers, such as orlistat or sibutramine; insulin sensitizers, such as thiazolidinediones (TZDs), metformin, and meglitinides; lipid lowering agents, such as statins, fibrates, and omega-3 fatty acids; antioxidants, such as vitamin E, betaine, N-acetyl-cysteine, lecithin, silymarin, and beta-carotene; anti-TNF agents, such as pentoxifylline; probiotics, such as VSL#3; and cytoprotectants, such as ursodeoxycholic acid (UDCA).Other suitable treatments include ACE inhibitors / ARBs, oligofructose, and incretin analogs.

[0051] Additional examples of liver disease therapeutics (e.g., for use in NASH) include OCALIVA® (obeticholic acid), selonsertib, elafibranor, cenicriviroc, GR_MD_02, MGL_3196, IMM124E, ARAMCHOL™ (arachidylamide cholanic acid), GS0976, emricasan, vorixibat, NGM282, G S9674, tropifexor, MN_001, LMB763, BI_1467335, MSDC_0602, PF_05221304, DF102, saroglitazar, BMS986036, lanifibranor, semaglutide, nitazoxanide, GRI_0621, EYP001, VK2809, nalmefene, LIK066, MT_3995, elobixibat, namodenoside Song, foralumab, SAR425899, sotagliflozin, EDP_305, isosabutate, gemcabene, TERN_101, KBP_042, PF_06865571, DUR928, PF_06835919, NGM313, BMS_986171, namacizumab, CER_209, ND_L02_s0201, RTU_1096, DR These include, but are not limited to, X_065, IONIS_DGAT2Rx, INT_767, NC_001, Seradelpal, PXL770, TERN_201, NV556, AZD2693, SP_1373, VK0214, Hepastem, TGFTX4, RLBN1127, GKT_137831, RYI_018, CB4209-CB4211, and JH_0920.

[0052] In some embodiments, the dosage of a therapeutic agent for treating or inhibiting liver disease can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for subjects who are heterozygous for a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3 compared to subjects who are CREB3L3 normative (i.e., receiving a lower standard dosage). In some embodiments, the dosage of a therapeutic agent for treating or inhibiting liver disease can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dosage of a therapeutic agent for treating or inhibiting liver disease in subjects who are heterozygous for a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3 can be administered less frequently compared to subjects who are CREB3L3 normative.

[0053] The administration of the therapeutic agent for treating or inhibiting liver disease and / or the CREB3L3 inhibitor 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 the same dose or 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 subject can be treated for an extended period of time, such as, for example, 6 months, 1 year, or more. Furthermore, the therapeutic agent for treating or inhibiting liver disease and / or the CREB3L3 inhibitor can be administered sequentially or simultaneously. Furthermore, the therapeutic agent for treating or inhibiting liver disease and / or the CREB3L3 inhibitor can be administered in separate compositions or together in the same composition.

[0054] Administration of the therapeutic agent and / or CREB3L3 inhibitor for treating or inhibiting liver disease can occur by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single dose for administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0055] As used herein, the terms "treat", "treating", and "treatment" and "prevent", "prevention", and "prevention" refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect, respectively. In some embodiments, the therapeutic effect includes one or more of the following after administration of the agent or a composition comprising the agent: reduction / reduction of liver disease, reduction / reduction of the severity of liver disease (e.g., reduction or inhibition of onset of liver disease), reduction / reduction of symptoms and liver disease-related effects, delaying the onset of symptoms and liver disease-related effects, reducing the severity of symptoms of liver disease-related effects, reducing the severity of acute episodes, reducing the number of symptoms and liver disease-related effects, shortening the latency period of symptoms and liver disease-related effects, ameliorating symptoms and liver disease-related effects, reducing secondary symptoms, reducing secondary infections, preventing recurrence of liver disease, reducing the number or frequency of recurrent episodes, increasing the latency period between symptomatic episodes, increasing the time to sustained progression, promoting remission, inducing remission, enhancing remission, accelerating recovery, or increasing the effectiveness or reducing resistance to alternative therapeutic agents, and / or increasing the survival time of the affected host animal. A prophylactic effect may include complete or partial avoidance / inhibition or delay (e.g., complete or partial avoidance / inhibition or delay) of the onset / progression of liver disease following administration of a treatment protocol, and increasing the survival time of an affected host animal. Treatment of liver disease includes treatment of subjects already diagnosed with some form of liver disease, either at a clinical stage or clinical symptoms, delaying the onset or progression or progression or worsening of symptoms or signs of liver disease, and / or preventing and / or reducing the severity of liver disease.

[0056] The present disclosure also provides a method for identifying a subject at high risk of developing liver disease. In some embodiments, the method includes determining or having determined the presence or absence of a CREB3L3 variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule and / or a cDNA molecule) encoding a CREB3L3 predicted loss-of-function polypeptide in a biological sample obtained from a subject. If a subject lacks a CREB3L3 variant nucleic acid molecule encoding a CREB3L3 predicted loss-of-function polypeptide (i.e., the subject is classified as a CREB3L3 standard by genotyping), the subject has a high risk of developing liver disease. If a subject has a CREB3L3 variant nucleic acid molecule encoding a CREB3L3 predicted loss-of-function polypeptide (i.e., the subject is heterozygous or homozygous for a CREB3L3 variant nucleic acid molecule), the subject has a lower risk of developing liver disease compared to a subject that is a CREB3L3 standard.

[0057] Having a single copy of a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3 further protects a subject from developing liver disease than not having a copy of a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3 (i.e., heterozygous for the CREB3L3 variant nucleic acid molecule) protects a subject from developing liver disease, and it is also believed that having two copies of a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3 (i.e., homozygous for the CREB3L3 variant nucleic acid molecule) can further protect a subject from developing liver disease compared to a subject having a single copy. Thus, in some embodiments, a single copy of the CREB3L3 variant nucleic acid molecule that encodes the loss-of-function polypeptide predicted by CREB3L3 may not completely protect a subject from developing liver disease, but may instead provide partial or incomplete protection.Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of liver disease that still exist in the subject that has a single copy of the CREB3L3 variant nucleic acid molecule that encodes the loss-of-function polypeptide predicted by CREB3L3, which may result in incomplete protection from developing liver disease.

[0058] Detecting the presence or absence of a CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in a biological sample from a subject and / or determining whether a subject has a CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in CREB3L3 can be performed by any of the methods described herein. In some embodiments, these methods can be 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 CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in CREB3L3 can be present in a cell obtained from the subject.

[0059] In some embodiments, once a subject is identified as having a high risk of developing liver disease, the subject is further treated with a therapeutic agent that treats or inhibits liver disease and / or a CREB3L3 inhibitor, as described herein. For example, if a subject is CREB3L3 based and therefore at high risk of developing liver disease, the subject is administered a CREB3L3 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent that treats or inhibits liver disease. In some embodiments, if the subject is heterozygous for a CREB3L3 variant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3, the subject is administered a therapeutic agent that treats or inhibits liver disease at a dose equal to or less than the standard dose, and is also administered a CREB3L3 inhibitor. In some embodiments, the subject is CREB3L3 based. In some embodiments, the subject is heterozygous for a CREB3L3 variant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3.

[0060] The present disclosure also provides a method for detecting the presence or absence of a CREB3L3 variant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3 in a biological sample obtained from a subject, and / or a CREB3L3 variant mRNA molecule encoding a loss-of-function polypeptide predicted by CREB3L3 in a biological sample obtained from a subject, and / or a CREB3L3 variant cDNA molecule encoding a loss-of-function polypeptide predicted by CREB3L3 generated from an mRNA molecule in a biological sample obtained from a subject. It is understood that gene sequences within a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms, such as single nucleotide polymorphisms (SNPs). The sequences provided herein for the CREB3L3 variant genomic nucleic acid molecule, the CREB3L3 variant mRNA molecule, and the CREB3L3 variant cDNA molecule are merely exemplary sequences. Other sequences for the CREB3L3 variant genomic nucleic acid molecule, the variant mRNA molecule, and the variant cDNA molecule are also possible.

[0061] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The biological sample may include any clinically relevant tissue, such as, for example, a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as, for example, blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the biological sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be subjected to different treatments depending on the assay employed. For example, when detecting CREB3L3 mutant nucleic acid molecules, a pretreatment designed to isolate or enrich the biological sample for CREB3L3 mutant nucleic acid molecules may be employed. For this purpose, various techniques may be used. When detecting the level of CREB3L3 mutant mRNA molecules, various techniques may be used to enrich the biological sample containing mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular mutant genomic DNA locus.

[0062] The present disclosure also provides a method for detecting a CREB3L3 mutant nucleic acid molecule or its complement that encodes a loss-of-function polypeptide predicted by CREB3L3 in a subject. The method includes assaying a biological sample obtained from a subject to determine whether a nucleic acid molecule in the biological sample is a CREB3L3 mutant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3.

[0063] In some embodiments, a CREB3L3 mutant nucleic acid molecule or its complement encoding a loss-of-function polypeptide predicted for CREB3L3 is a genomic nucleic acid molecule having a nucleotide sequence including an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, or its complement.

[0064] In some embodiments, a CREB3L3 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CREB3L3, or its complement, has an adenine at a position corresponding to 661 in SEQ ID NO: 17, or its complement; an adenine at a position corresponding to 649 in SEQ ID NO: 18, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO: 19, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO: 20, or its complement; an adenine at a position corresponding to 658 in SEQ ID NO: 21, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO: 22, or its complement; an adenine, or its complement, at a position corresponding to position 661 set forth in SEQ ID NO:23; an adenine, or its complement, at a position corresponding to position 663 set forth in SEQ ID NO:24; an adenine, or its complement, at a position corresponding to position 660 set forth in SEQ ID NO:25; an adenine, or its complement, at a position corresponding to position 649 set forth in SEQ ID NO:26; an adenine, or its complement, at a position corresponding to position 624 set forth in SEQ ID NO:27; or an adenine, or its complement, at a position corresponding to position 691 set forth in SEQ ID NO:28.

[0065] In some embodiments, a CREB3L3 mutant nucleic acid molecule encoding a predicted loss-of-function polypeptide of CREB3L3, or its complement, has an adenine at a position corresponding to 661 in SEQ ID NO:45, or its complement; an adenine at a position corresponding to 649 in SEQ ID NO:46, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:47, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:48, or its complement; an adenine at a position corresponding to 658 in SEQ ID NO:49, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO:50, or its complement; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:51, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or its complement.

[0066] In some embodiments, the CREB3L3 variant nucleic acid molecule contains: i) an adenine at a position corresponding to position 6,120 set forth in SEQ ID NO:2 (for a genomic nucleic acid molecule); ii) an adenine at a position corresponding to position 661 set forth in SEQ ID NO:17, position 649 set forth in SEQ ID NO:18, position 624 set forth in SEQ ID NO:19, position 624 set forth in SEQ ID NO:20, position 658 set forth in SEQ ID NO:21, position 661 set forth in SEQ ID NO:22, position 661 set forth in SEQ ID NO:23, position 663 set forth in SEQ ID NO:24, position 660 set forth in SEQ ID NO:25, position 649 set forth in SEQ ID NO:26, position 624 set forth in SEQ ID NO:27, or position 691 set forth in SEQ ID NO:28. or iii) has a nucleotide sequence comprising an adenine (for a cDNA molecule derived from an mRNA molecule) at a position corresponding to: position 661 set forth in SEQ ID NO: 45, position 649 set forth in SEQ ID NO: 46, position 624 set forth in SEQ ID NO: 47, position 624 set forth in SEQ ID NO: 48, position 658 set forth in SEQ ID NO: 49, position 661 set forth in SEQ ID NO: 50, position 661 set forth in SEQ ID NO: 51, position 663 set forth in SEQ ID NO: 52, position 660 set forth in SEQ ID NO: 53, position 649 set forth in SEQ ID NO: 54, position 624 set forth in SEQ ID NO: 55, or position 691 set forth in SEQ ID NO: 56.

[0067] In some embodiments, the biological sample comprises cells or cell lysates. Such methods can further comprise, for example, obtaining a biological sample from a subject that comprises a CREB3L3 genomic nucleic acid molecule or an mRNA molecule, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can comprise, for example, determining the identity of these positions of a particular CREB3L3 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0068] In some embodiments, the determining, detecting, or sequence analysis includes sequencing at least a portion of the nucleotide sequence of a CREB3L3 genomic nucleic acid molecule, a CREB3L3 mRNA molecule, or a CREB3L3 cDNA molecule generated from the mRNA molecule in a biological sample, wherein the sequenced portion contains one or more mutations that cause or are predicted to cause loss of function (partial or complete).

[0069] In some embodiments, the determining, detecting, or sequence analyzing step comprises: i) sequencing at least a portion of the nucleotide sequence of a CREB3L3 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 6,120 of SEQ ID NO:2, or a complement thereof; ii) detecting at least a portion of the nucleotide sequence of a CREB3L3 genomic nucleic acid molecule in the biological sample; and / or iii) sequencing at least a portion of the nucleotide sequence of the mRNA molecule, wherein the sequenced portion comprises a position corresponding to: position 661 in SEQ ID NO: 17, or a complement thereof; position 649 in SEQ ID NO: 18, or a complement thereof; position 624 in SEQ ID NO: 19, or a complement thereof; position 624 in SEQ ID NO: 20, or a complement thereof; position 658 in SEQ ID NO: 21, or a complement thereof; position 661 in SEQ ID NO: 22, or a complement thereof; position 661 in SEQ ID NO: 23, or a complement thereof; position 663 in SEQ ID NO: 24, or a complement thereof; position 660 in SEQ ID NO: 25, or a complement thereof; position 649 in SEQ ID NO: 26, or a complement thereof; position 624 in SEQ ID NO: 27, or a complement thereof; or position 691 in SEQ ID NO: 28, or a complement thereof; and / or iii) sequencing at least a portion of the nucleotide sequence of the mRNA molecule, wherein the sequenced portion comprises a position corresponding to: position 661 in SEQ ID NO: 17, or a complement thereof; position 649 in SEQ ID NO: 26, or a complement thereof; position 624 in SEQ ID NO: 27, or a complement thereof; or position 691 in SEQ ID NO: 28, or a complement thereof; the step of sequencing at least a portion of the nucleotide sequence of the cDNA molecule, wherein the sequenced portion comprises a position corresponding to: position 661 set forth in SEQ ID NO: 45, or its complement; position 649 set forth in SEQ ID NO: 46, or its complement; position 624 set forth in SEQ ID NO: 47, or its complement; position 624 set forth in SEQ ID NO: 48, or its complement; position 658 set forth in SEQ ID NO: 49, or its complement; position 661 set forth in SEQ ID NO: 50, or its complement; position 661 set forth in SEQ ID NO: 51, or its complement; position 663 set forth in SEQ ID NO: 52, or its complement; position 660 set forth in SEQ ID NO: 53, or its complement; position 649 set forth in SEQ ID NO: 54, or its complement; position 624 set forth in SEQ ID NO: 55, or its complement; or position 691 set forth in SEQ ID NO: 56, or its complement.A sequenced portion of the CREB3L3 nucleic acid molecule in the biological sample contains an adenine at a position corresponding to position 6,120 set forth in SEQ ID NO:2; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:17, position 649 set forth in SEQ ID NO:18, position 624 set forth in SEQ ID NO:19, position 624 set forth in SEQ ID NO:20, position 658 set forth in SEQ ID NO:21, position 661 set forth in SEQ ID NO:22, position 661 set forth in SEQ ID NO:23, position 663 set forth in SEQ ID NO:24, position 660 set forth in SEQ ID NO:25, position 649 set forth in SEQ ID NO:26, position 624 set forth in SEQ ID NO:27, or position 691 set forth in SEQ ID NO:28; or an adenine at a position corresponding to position 661 set forth in SEQ ID NO:17, position 649 set forth in SEQ ID NO:18, position 624 set forth in SEQ ID NO:19, position 624 set forth in SEQ ID NO:20, position 658 set forth in SEQ ID NO:21, position 661 set forth in SEQ ID NO:22, position 661 set forth in SEQ ID NO:23, position 663 set forth in SEQ ID NO:24, position 660 set forth in SEQ ID NO:25, position 649 set forth in SEQ ID NO:26, position 624 set forth in SEQ ID NO:27, or position 691 set forth in SEQ ID NO:28; position 661 set forth in SEQ ID NO: 46, position 649 set forth in SEQ ID NO: 46, position 624 set forth in SEQ ID NO: 47, position 624 set forth in SEQ ID NO: 48, position 658 set forth in SEQ ID NO: 49, position 661 set forth in SEQ ID NO: 50, position 661 set forth in SEQ ID NO: 51, position 663 set forth in SEQ ID NO: 52, position 660 set forth in SEQ ID NO: 53, position 649 set forth in SEQ ID NO: 54, position 624 set forth in SEQ ID NO: 55, or position 691 set forth in SEQ ID NO: 56, the CREB3L3 nucleic acid molecule in the biological sample is a CREB3L3 mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted for CREB3L3.

[0070] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a CREB3L3 genomic nucleic acid molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to position 6,120 of SEQ ID NO:2, or a complement thereof. If the sequenced portion of the CREB3L3 nucleic acid molecule in the biological sample comprises an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, then the CREB3L3 nucleic acid molecule in the biological sample is a CREB3L3 mutant genomic nucleic acid molecule that encodes a loss-of-function polypeptide predicted for CREB3L3.

[0071] In some embodiments, the determining, detecting or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the CREB3L3 mRNA molecule in the biological sample, wherein the sequenced portion comprises a position corresponding to: position 661 set forth in SEQ ID NO: 17, or its complement; position 649 set forth in SEQ ID NO: 18, or its complement; position 624 set forth in SEQ ID NO: 19, or its complement; position 624 set forth in SEQ ID NO: 20, or its complement; position 658 set forth in SEQ ID NO: 21, or its complement; position 661 set forth in SEQ ID NO: 22, or its complement; position 661 set forth in SEQ ID NO: 23, or its complement; position 663 set forth in SEQ ID NO: 24, or its complement; position 660 set forth in SEQ ID NO: 25, or its complement; position 649 set forth in SEQ ID NO: 26, or its complement; position 624 set forth in SEQ ID NO: 27, or its complement; or position 691 set forth in SEQ ID NO: 28, or its complement. If the sequenced portion of the CREB3L3 mRNA molecule in the biological sample contains an adenine at a position corresponding to position 661 of SEQ ID NO:17, position 649 of SEQ ID NO:18, position 624 of SEQ ID NO:19, position 624 of SEQ ID NO:20, position 658 of SEQ ID NO:21, position 661 of SEQ ID NO:22, position 661 of SEQ ID NO:23, position 663 of SEQ ID NO:24, position 660 of SEQ ID NO:25, position 649 of SEQ ID NO:26, position 624 of SEQ ID NO:27, or position 691 of SEQ ID NO:28, then the CREB3L3 nucleic acid molecule in the biological sample is a CREB3L3 mutant mRNA molecule encoding a loss-of-function polypeptide predicted for CREB3L3.

[0072] In some embodiments, the determining, detecting, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of a CREB3L3 cDNA molecule generated from an mRNA molecule in a biological sample, wherein the sequenced portion comprises positions corresponding to: position 661 set forth in SEQ ID NO: 45, or its complement; position 649 set forth in SEQ ID NO: 46, or its complement; position 624 set forth in SEQ ID NO: 47, or its complement; position 624 set forth in SEQ ID NO: 48, or its complement; position 658 set forth in SEQ ID NO: 49, or its complement; position 661 set forth in SEQ ID NO: 50, or its complement; position 661 set forth in SEQ ID NO: 51, or its complement; position 663 set forth in SEQ ID NO: 52, or its complement; position 660 set forth in SEQ ID NO: 53, or its complement; position 649 set forth in SEQ ID NO: 54, or its complement; position 624 set forth in SEQ ID NO: 55, or its complement; position 691 set forth in SEQ ID NO: 56, or its complement. If the sequenced portion of the CREB3L3 cDNA molecule in the biological sample contains an adenine at a position corresponding to position 661 set forth in SEQ ID NO:45, position 649 set forth in SEQ ID NO:46, position 624 set forth in SEQ ID NO:47, position 624 set forth in SEQ ID NO:48, position 658 set forth in SEQ ID NO:49, position 661 set forth in SEQ ID NO:50, position 661 set forth in SEQ ID NO:51, position 663 set forth in SEQ ID NO:52, position 660 set forth in SEQ ID NO:53, position 649 set forth in SEQ ID NO:54, position 624 set forth in SEQ ID NO:55, or position 691 set forth in SEQ ID NO:56, then the CREB3L3 nucleic acid molecule in the biological sample is a CREB3L3 mutant cDNA molecule encoding a loss-of-function polypeptide predicted for CREB3L3.

[0073] In some embodiments, the determining, detecting or sequence analyzing step comprises determining, detecting or sequencing a) a CREB3L3 genomic nucleic acid molecule adjacent to i) a position corresponding to position 6,120 set forth in SEQ ID NO:2, or its complement; ii) a CREB3L3 genomic nucleic acid molecule adjacent to a position corresponding to position 661 set forth in SEQ ID NO:17, or its complement; position 649 set forth in SEQ ID NO:18, or its complement; position 624 set forth in SEQ ID NO:19, or its complement; position 624 set forth in SEQ ID NO:20, or its complement; position 658 set forth in SEQ ID NO:21, or its complement; position 661 set forth in SEQ ID NO:22, or its complement; position 661 set forth in SEQ ID NO:23, or its complement; position 663 set forth in SEQ ID NO:24, or its complement; position 660 set forth in SEQ ID NO:25, or its complement; position 649 set forth in SEQ ID NO:26, or its complement; position 624 set forth in SEQ ID NO:27, or its complement; or position 691 set forth in SEQ ID NO:28. an mRNA molecule or its complement; and / or iii) a CREB3L3 molecule adjacent to a position corresponding to position 661 as set forth in SEQ ID NO: 45, or its complement; position 649 as set forth in SEQ ID NO: 46, or its complement; position 624 as set forth in SEQ ID NO: 47, or its complement; position 624 as set forth in SEQ ID NO: 48, or its complement; position 658 as set forth in SEQ ID NO: 49, or its complement; position 661 as set forth in SEQ ID NO: 50, or its complement; position 661 as set forth in SEQ ID NO: 51, or its complement; position 663 as set forth in SEQ ID NO: 52, or its complement; position 660 as set forth in SEQ ID NO: 53, or its complement; position 649 as set forth in SEQ ID NO: 54, or its complement; or position 624 as set forth in SEQ ID NO: 55, or its complement; position 691 as set forth in SEQ ID NO: 56. contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule or its complement; b) i) a CREB3L3 genomic nucleic acid molecule corresponding to position 6,120 set forth in SEQ ID NO:2, or its complement; ii) position 661 set forth in SEQ ID NO:17, or its complement; position 649 set forth in SEQ ID NO:18, or its complement; position 624 set forth in SEQ ID NO:19, or its complement; position 624 set forth in SEQ ID NO:20, or its complement; position 658 set forth in SEQ ID NO:21, or its complement;CREB3L3 corresponding to position 661 in SEQ ID NO:22, or its complement; position 661 in SEQ ID NO:23, or its complement; position 663 in SEQ ID NO:24, or its complement; position 660 in SEQ ID NO:25, or its complement; position 649 in SEQ ID NO:26, or its complement; position 624 in SEQ ID NO:27, or its complement; or position 691 in SEQ ID NO:28, or its complement. an mRNA molecule or its complement; and / or iii) a CREB3L3 corresponding to position 661 as set forth in SEQ ID NO: 45, or its complement; position 649 as set forth in SEQ ID NO: 46, or its complement; position 624 as set forth in SEQ ID NO: 47, or its complement; position 624 as set forth in SEQ ID NO: 48, or its complement; position 658 as set forth in SEQ ID NO: 49, or its complement; position 661 as set forth in SEQ ID NO: 50, or its complement; position 661 as set forth in SEQ ID NO: 51, or its complement; position 663 as set forth in SEQ ID NO: 52, or its complement; position 660 as set forth in SEQ ID NO: 53, or its complement; position 649 as set forth in SEQ ID NO: 54, or its complement; position 624 as set forth in SEQ ID NO: 55, or its complement; or position 691 as set forth in SEQ ID NO: 56. extending the primer at least through a position of the nucleotide sequence of the cDNA molecule or its complement; c) determining that the extension product of the primer is: an adenine at a position corresponding to position 6,120 in SEQ ID NO:2, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:17, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:18, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:19, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:25, or its complement;an adenine at a position corresponding to position 649 in SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 in SEQ ID NO:28, or its complement; or an adenine at a position corresponding to position 661 in SEQ ID NO:45, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:46, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:47, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:48, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:49 or a complement thereof; an adenine at a position corresponding to 661 in SEQ ID NO:50, or a complement thereof; an adenine at a position corresponding to 661 in SEQ ID NO:51, or a complement thereof; an adenine at a position corresponding to 663 in SEQ ID NO:52, or a complement thereof; an adenine at a position corresponding to 660 in SEQ ID NO:53, or a complement thereof; an adenine at a position corresponding to 649 in SEQ ID NO:54, or a complement thereof; an adenine at a position corresponding to 624 in SEQ ID NO:55, or a complement thereof; or an adenine at a position corresponding to 691 in SEQ ID NO:56, or a complement thereof;

[0074] In some embodiments, the determining, detecting, or sequence analyzing step includes: a) contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of a CREB3L3 genomic nucleic acid molecule or its complement adjacent to a position corresponding to position 6,120 set forth in SEQ ID NO:2, or its complement; b) extending the primer at least through the position of the nucleotide sequence of a CREB3L3 genomic nucleic acid molecule or its complement corresponding to position 6,120 set forth in SEQ ID NO:2, or its complement; and c) determining whether the extension product of the primer contains an adenine at the position corresponding to position 6,120 set forth in SEQ ID NO:2, or its complement.

[0075] In some embodiments, the determining, detecting or sequence analyzing step is performed by: a) detecting a CREB3L3 gene adjacent to a position corresponding to: position 661 set forth in SEQ ID NO: 17, or its complement; position 649 set forth in SEQ ID NO: 18, or its complement; position 624 set forth in SEQ ID NO: 19, or its complement; position 624 set forth in SEQ ID NO: 20, or its complement; position 658 set forth in SEQ ID NO: 21, or its complement; position 661 set forth in SEQ ID NO: 22, or its complement; position 661 set forth in SEQ ID NO: 23, or its complement; position 663 set forth in SEQ ID NO: 24, or its complement; position 660 set forth in SEQ ID NO: 25, or its complement; position 649 set forth in SEQ ID NO: 26, or its complement; position 624 set forth in SEQ ID NO: 27, or its complement; or position 691 set forth in SEQ ID NO: 28, or its complement. contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the mRNA molecule or its complement; and b) detecting a CREB3L3 gene corresponding to position 661 of SEQ ID NO: 17, or its complement; position 649 of SEQ ID NO: 18, or its complement; position 624 of SEQ ID NO: 19, or its complement; position 624 of SEQ ID NO: 20, or its complement; position 658 of SEQ ID NO: 21, or its complement; position 661 of SEQ ID NO: 22, or its complement; position 661 of SEQ ID NO: 23, or its complement; position 663 of SEQ ID NO: 24, or its complement; position 660 of SEQ ID NO: 25, or its complement; position 649 of SEQ ID NO: 26, or its complement; position 624 of SEQ ID NO: 27, or its complement; or position 691 of SEQ ID NO: 28, or its complement. extending the primer through at least a position in the nucleotide sequence of the mRNA molecule; and c) determining that the extension product of the primer is: an adenine at a position corresponding to 661 in SEQ ID NO:17, or its complement; an adenine at a position corresponding to 649 in SEQ ID NO:18, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:19, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to 658 in SEQ ID NO:21, or its complement;and determining whether the nucleic acid sequence contains an adenine, or its complement, at a position corresponding to 661 in SEQ ID NO:22; an adenine, or its complement, at a position corresponding to 661 in SEQ ID NO:23; an adenine, or its complement, at a position corresponding to 663 in SEQ ID NO:24; an adenine, or its complement, at a position corresponding to 660 in SEQ ID NO:25; an adenine, or its complement, at a position corresponding to 649 in SEQ ID NO:26; an adenine, or its complement, at a position corresponding to 624 in SEQ ID NO:27; or an adenine, or its complement, at a position corresponding to 691 in SEQ ID NO:28.

[0076] In some embodiments, the determining, detecting or sequence analyzing step comprises: a) detecting a CREB3L3 gene adjacent to a position corresponding to: position 661 set forth in SEQ ID NO: 45, or its complement; position 649 set forth in SEQ ID NO: 46, or its complement; position 624 set forth in SEQ ID NO: 47, or its complement; position 624 set forth in SEQ ID NO: 48, or its complement; position 658 set forth in SEQ ID NO: 49, or its complement; position 661 set forth in SEQ ID NO: 50, or its complement; position 661 set forth in SEQ ID NO: 51, or its complement; position 663 set forth in SEQ ID NO: 52, or its complement; position 660 set forth in SEQ ID NO: 53, or its complement; position 649 set forth in SEQ ID NO: 54, or its complement; position 624 set forth in SEQ ID NO: 55, or its complement; or position 691 set forth in SEQ ID NO: 56, or its complement. contacting the biological sample with a primer that hybridizes to a portion of the nucleotide sequence of the cDNA molecule or its complement; and b) detecting a CREB3L3 gene corresponding to position 661 of SEQ ID NO: 45, or its complement; position 649 of SEQ ID NO: 46, or its complement; position 624 of SEQ ID NO: 47, or its complement; position 624 of SEQ ID NO: 48, or its complement; position 658 of SEQ ID NO: 49, or its complement; position 661 of SEQ ID NO: 50, or its complement; position 661 of SEQ ID NO: 51, or its complement; position 663 of SEQ ID NO: 52, or its complement; position 660 of SEQ ID NO: 53, or its complement; position 649 of SEQ ID NO: 54, or its complement; position 624 of SEQ ID NO: 55, or its complement; or position 691 of SEQ ID NO: 56, or its complement. extending the primer through at least a position in the nucleotide sequence of the cDNA molecule; and c) determining whether the extension product of the primer is: an adenine at a position corresponding to 661 in SEQ ID NO:45, or its complement; an adenine at a position corresponding to 649 in SEQ ID NO:46, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:47, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:48, or its complement; an adenine at a position corresponding to 658 in SEQ ID NO:49, or its complement;and determining whether the nucleic acid sequence contains an adenine at a position corresponding to 661 in SEQ ID NO:50, or a complement thereof; an adenine at a position corresponding to 661 in SEQ ID NO:51, or a complement thereof; an adenine at a position corresponding to 663 in SEQ ID NO:52, or a complement thereof; an adenine at a position corresponding to 660 in SEQ ID NO:53, or a complement thereof; an adenine at a position corresponding to 649 in SEQ ID NO:54, or a complement thereof; an adenine at a position corresponding to 624 in SEQ ID NO:55, or a complement thereof; or an adenine at a position corresponding to 691 in SEQ ID NO:56, or a complement thereof.

[0077] In some embodiments, the entire nucleic acid molecule is sequenced. In some embodiments, only the CREB3L3 genomic nucleic acid molecule is analyzed. In some embodiments, only the CREB3L3 mRNA is analyzed. In some embodiments, only the CREB3L3 cDNA obtained from the CREB3L3 mRNA is analyzed.

[0078] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of a CREB3L3 nucleic acid molecule or its complement in the biological sample, wherein the amplified portion contains an adenine at a position corresponding to position 6,120 set forth in SEQ ID NO:2, or its complement; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:17, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:18, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:19, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:26, or its complement; or an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:27; or an adenine, or its complement, at a position corresponding to position 691 in SEQ ID NO:28; or an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:45; an adenine, or its complement, at a position corresponding to position 649 in SEQ ID NO:46; an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:47; or an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:48 adenine, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:49, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:50, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:51, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:54, or its complement;a) amplifying the nucleic acid molecule containing an adenine, or its complement, at a position corresponding to position 624 of SEQ ID NO:55; or an adenine, or its complement, at a position corresponding to position 691 of SEQ ID NO:56; b) labeling the amplified nucleic acid molecule with a detectable label; c) coupling the labeled nucleic acid molecule to a mutation-specific probe comprising an adenine, or its complement, at a position corresponding to position 6,120 of SEQ ID NO:2; an adenine, or its complement, at a position corresponding to position 661 of SEQ ID NO:17; or an adenine, or its complement, at a position corresponding to position 649 of SEQ ID NO:18. an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:19; an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:20; an adenine, or its complement, at a position corresponding to position 658 in SEQ ID NO:21; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:22; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:23; an adenine, or its complement, at a position corresponding to position 663 in SEQ ID NO:24; an adenine, or its complement, at a position corresponding to position 664 in SEQ ID NO:25; or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 in SEQ ID NO:28, or its complement; or an adenine at a position corresponding to position 661 in SEQ ID NO:45, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:46, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:47, or its complement. an adenine at a position corresponding to position 624 in SEQ ID NO:48, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:49, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:50, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:51, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:53, or its complement;a) contacting said support with said mutation-specific probe, said mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions with a nucleic acid sequence of the amplified nucleic acid molecule comprising an adenine at a position corresponding to 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to 691 set forth in SEQ ID NO:56, or its complement; and d) detecting the detectable label.

[0079] In some embodiments, the determining, detecting, or sequence analyzing step includes: a) amplifying at least a portion of a CREB3L3 genomic nucleic acid molecule or its complement in a biological sample, the portion comprising an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, or its complement; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising a mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, or its complement; and d) detecting the detectable label.

[0080] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of a CREB3L3 mRNA molecule or its complement in the biological sample, the portion comprising an adenine at a position corresponding to position 661 of SEQ ID NO: 17, or its complement; an adenine at a position corresponding to position 649 of SEQ ID NO: 18, or its complement; an adenine at a position corresponding to position 624 of SEQ ID NO: 19, or its complement; an adenine at a position corresponding to position 624 of SEQ ID NO: 20, or its complement; an adenine at a position corresponding to position 658 of SEQ ID NO: 21, or its complement; an adenine at a position corresponding to position 658 of SEQ ID NO: 22, or its complement; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 69 a) amplifying said nucleic acid molecule with a detectable label; b) labeling said amplified nucleic acid molecule with a detectable label; c) comparing said labeled nucleic acid molecule with a mutation-specific probe comprising an adenine at a position corresponding to position 661 in SEQ ID NO:17, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:18, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:19, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:26, or its complement;a) contacting the support with the mutation-specific probe, the mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising an adenine at a position corresponding to position 624 set forth in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:28, or its complement; and d) detecting the detectable label.

[0081] In some embodiments, the determining, detecting, or sequence analyzing step comprises: a) amplifying at least a portion of a CREB3L3 cDNA molecule or its complement in the biological sample, the portion being an adenine at a position corresponding to position 661 of SEQ ID NO:45, or its complement; an adenine at a position corresponding to position 649 of SEQ ID NO:46, or its complement; an adenine at a position corresponding to position 624 of SEQ ID NO:47, or its complement; an adenine at a position corresponding to position 624 of SEQ ID NO:48, or its complement; an adenine at a position corresponding to position 658 of SEQ ID NO:49, or its complement; an adenine at a position corresponding to position 658 of SEQ ID NO:50, or its complement; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:51, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 69 a) amplifying said nucleic acid molecule with a detectable label; b) labeling said amplified nucleic acid molecule with a detectable label; c) comparing said labeled nucleic acid molecule with a mutation-specific probe comprising an adenine at a position corresponding to position 661 in SEQ ID NO:45, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:46, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:47, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:48, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:49, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:50, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:51, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:54, or its complement;a) contacting said support with said mutation-specific probe, said mutation-specific probe comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of the amplified nucleic acid molecule comprising an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or its complement; and d) detecting the detectable label.

[0082] In some embodiments, the nucleic acid molecule is mRNA and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step. In some embodiments, the determining, detecting, or sequence analyzing step comprises: contacting a CREB3L3 nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe detects an adenine at a position corresponding to position 6,120 set forth in SEQ ID NO:2, or its complement; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:17, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:18, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:19, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:26, or its complement; or adenine, or its complement, at a position corresponding to position 649 in SEQ ID NO:27; or an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:28; or an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:45; an adenine, or its complement, at a position corresponding to position 649 in SEQ ID NO:46; an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:47; or an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:48 an adenine, or its complement, at a position corresponding to position 658 in SEQ ID NO:49; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:50; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:51; an adenine, or its complement, at a position corresponding to position 663 in SEQ ID NO:52; an adenine, or its complement, at a position corresponding to position 660 in SEQ ID NO:53; an adenine, or its complement, at a position corresponding to position 649 in SEQ ID NO:54;the contacting comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a CREB3L3 nucleic acid molecule or its complement that comprises an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or a complement thereof; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or a complement thereof, and detecting the detectable label;

[0083] In some embodiments, the determining step, detecting step, or sequence analysis includes: contacting a CREB3L3 genomic nucleic acid molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of a CREB3L3 genomic nucleic acid molecule or its complement that comprises an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, or its complement, and detecting the detectable label.

[0084] In some embodiments, the determining, detecting, or sequence analyzing step comprises: contacting a CREB3L3 mRNA molecule or its complement in a biological sample with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe detects an adenine at a position corresponding to position 661 in SEQ ID NO: 17, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO: 18, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO: 19, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO: 20, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO: 21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO: 22, or its complement; the contacting comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of a CREB3L3 mRNA molecule or its complement comprising: adenine, or its complement; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:28, or its complement; and detecting the detectable label.

[0085] In some embodiments, the determining, detecting, or sequence analyzing step comprises: contacting a CREB3L3 cDNA molecule generated from an mRNA molecule in a biological sample, or its complement, with a mutation-specific probe comprising a detectable label, wherein the mutation-specific probe detects an adenine at a position corresponding to position 661 in SEQ ID NO:45, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:46, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:47, or its complement; an adenine at a position corresponding to position 624 in SEQ ID NO:48, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:49, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:50, or its complement; the contacting comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of a CREB3L3 cDNA molecule or its complement, the nucleotide sequence comprising: adenine, or its complement; an adenine at a position corresponding to position 661 set forth in SEQ ID NO:51, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or its complement; and detecting the detectable label.

[0086] In some embodiments, the CREB3L3 nucleic acid molecule is present in a cell obtained from the subject. Mutation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleic acid sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0087] In some embodiments, the determining, detecting, or sequence analysis step includes contacting the biological sample with a primer or probe, such as a mutation-specific primer or a mutation-specific probe, that specifically hybridizes to a CREB3L3 mutant genomic sequence, mutant mRNA sequence, or mutant cDNA sequence, but not to a corresponding CREB3L3 reference sequence, under stringent conditions, and determining whether hybridization has occurred.

[0088] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0089] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides including mutant genomic nucleic acid molecules, mutant mRNA molecules, or mutant cDNA molecules of CREB3L3. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length that is 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 highly stringent hybridization conditions. The probes and primers can have complete nucleotide sequence identity to consecutive nucleotides within the target nucleotide sequence, although probes that are different from the target nucleotide sequence and retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The probes and primers can 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 to the nucleotide sequence of the target nucleic acid molecule.

[0090] In some embodiments, a CREB3L3 nucleic acid molecule (genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) or its complement in a biological sample contains an adenine at a position corresponding to position 6,120 of SEQ ID NO:2; position 661 of SEQ ID NO:17, position 649 of SEQ ID NO:18, position 624 of SEQ ID NO:19, position 624 of SEQ ID NO:20, position 658 of SEQ ID NO:21, position 661 of SEQ ID NO:22, position 661 of SEQ ID NO:23, position 663 of SEQ ID NO:24, position 660 of SEQ ID NO:25, position 649 of SEQ ID NO:26, position 661 of SEQ ID NO:27, position 662 of SEQ ID NO:29, position 663 of SEQ ID NO:30, position 660 of SEQ ID NO:31, position 662 of SEQ ID NO:32, position 663 of SEQ ID NO:33, position 664 of SEQ ID NO:34, position 665 of SEQ ID NO:35, position 666 of SEQ ID NO:36, position 669 of SEQ ID NO:37, position 667 of SEQ ID NO:38, position 669 of SEQ ID NO:39, position 670 of SEQ ID NO:40, position 671 of SEQ ID NO:41, position 672 of SEQ ID NO:42, position 673 of SEQ ID NO:43, position 674 of SEQ ID NO:44, position 675 of SEQ ID NO:45, position 676 of SEQ ID NO:46, position 677 of SEQ ID NO:47, position 678 of SEQ ID NO:48, position 679 of SEQ ID NO:49, position or a nucleotide sequence containing an adenine at a position corresponding to position 624 set forth in SEQ ID NO: 7, or position 691 set forth in SEQ ID NO: 28; or a nucleotide sequence containing an adenine at a position corresponding to position 661 set forth in SEQ ID NO: 45, position 649 set forth in SEQ ID NO: 46, position 624 set forth in SEQ ID NO: 47, position 624 set forth in SEQ ID NO: 48, position 658 set forth in SEQ ID NO: 49, position 661 set forth in SEQ ID NO: 50, position 661 set forth in SEQ ID NO: 51, position 663 set forth in SEQ ID NO: 52, position 660 set forth in SEQ ID NO: 53, position 649 set forth in SEQ ID NO: 54, position 624 set forth in SEQ ID NO: 55, or position 691 set forth in SEQ ID NO: 56. To determine whether the nucleic acid sequence contains a nucleotide sequence, an adenine at a position corresponding to position 6,120 of SEQ ID NO:2; an adenine at a position corresponding to position 661 of SEQ ID NO:17, position 649 of SEQ ID NO:18, position 624 of SEQ ID NO:19, position 624 of SEQ ID NO:20, position 658 of SEQ ID NO:21, position 661 of SEQ ID NO:22, position 661 of SEQ ID NO:23, position 663 of SEQ ID NO:24, position 660 of SEQ ID NO:25, position 649 of SEQ ID NO:26, position 624 of SEQ ID NO:27, or position 691 of SEQ ID NO:28; or a first primer derived from the 5' flanking sequence adjacent to an adenine at a position corresponding to position 661 set forth in sequence number 45, position 649 set forth in SEQ ID NO:46, position 624 set forth in SEQ ID NO:47, position 624 set forth in SEQ ID NO:48, position 658 set forth in SEQ ID NO:49, position 661 set forth in SEQ ID NO:50, position 661 set forth in SEQ ID NO:51, position 663 set forth in SEQ ID NO:52, position 660 set forth in SEQ ID NO:53, position 649 set forth in SEQ ID NO:54, position 624 set forth in SEQ ID NO:55, or position 691 set forth in SEQ ID NO:56, and an adenine at a position corresponding to position 6,120 set forth in SEQ ID NO:2;an adenine at a position corresponding to position 661 of SEQ ID NO:17, position 649 of SEQ ID NO:18, position 624 of SEQ ID NO:19, position 624 of SEQ ID NO:20, position 658 of SEQ ID NO:21, position 661 of SEQ ID NO:22, position 661 of SEQ ID NO:23, position 663 of SEQ ID NO:24, position 660 of SEQ ID NO:25, position 649 of SEQ ID NO:26, position 624 of SEQ ID NO:27, or position 691 of SEQ ID NO:28; or an adenine at a position corresponding to position 661 of SEQ ID NO:45, position 649 of SEQ ID NO:46, position 624 of SEQ ID NO:47, position 624 of SEQ ID NO:48, position 658 of SEQ ID NO:49, position 661 of SEQ ID NO:50, position 661 of SEQ ID NO:51, position 663 of SEQ ID NO:52, position 660 of SEQ ID NO:53, and a second primer derived from a 3' flanking sequence adjacent to an adenine at a position corresponding to position 6,120 in SEQ ID NO:2; an adenine at a position corresponding to position 661 in SEQ ID NO:17, position 649 in SEQ ID NO:18, position 624 in SEQ ID NO:19, position 624 in SEQ ID NO:20, position 658 in SEQ ID NO:21, position 661 in SEQ ID NO:22, position 661 in SEQ ID NO:23, position 663 in SEQ ID NO:24, position 660 in SEQ ID NO:25, position 649 in SEQ ID NO:26, position 624 in SEQ ID NO:27, or position 691 in SEQ ID NO:28;or an amplicon can be produced that indicates the presence of a SNP at a position that encodes an adenine at a position corresponding to position 661 of SEQ ID NO: 45, position 649 of SEQ ID NO: 46, position 624 of SEQ ID NO: 47, position 624 of SEQ ID NO: 48, position 658 of SEQ ID NO: 49, position 661 of SEQ ID NO: 50, position 661 of SEQ ID NO: 51, position 663 of SEQ ID NO: 52, position 660 of SEQ ID NO: 53, position 649 of SEQ ID NO: 54, position 624 of SEQ ID NO: 55, or position 691 of SEQ ID NO: 56. In some embodiments, the length of the amplicon can range from a combination of the length of the primer pair plus one nucleotide base pair to any length of an amplicon that can be produced by a DNA amplification protocol. This distance can range from one nucleotide base pair to the limit of the amplification reaction, or up to about 20,000 nucleotide base pairs. Optionally, the primer pair is an adenine at a position corresponding to position 6,120 in SEQ ID NO:2; an adenine at a position corresponding to position 661 in SEQ ID NO:17, position 649 in SEQ ID NO:18, position 624 in SEQ ID NO:19, position 624 in SEQ ID NO:20, position 658 in SEQ ID NO:21, position 661 in SEQ ID NO:22, position 661 in SEQ ID NO:23, position 663 in SEQ ID NO:24, position 660 in SEQ ID NO:25, position 649 in SEQ ID NO:26, position 624 in SEQ ID NO:27, or position 691 in SEQ ID NO:28. or a position including an adenine at a position corresponding to position 661 of SEQ ID NO:45, position 649 of SEQ ID NO:46, position 624 of SEQ ID NO:47, position 624 of SEQ ID NO:48, position 658 of SEQ ID NO:49, position 661 of SEQ ID NO:50, position 661 of SEQ ID NO:51, position 663 of SEQ ID NO:52, position 660 of SEQ ID NO:53, position 649 of SEQ ID NO:54, position 624 of SEQ ID NO:55, or position 691 of SEQ ID NO:56, and an adenine at a position corresponding to position 6,120 of SEQ ID NO:2;an adenine at a position corresponding to position 661 of SEQ ID NO:17, position 649 of SEQ ID NO:18, position 624 of SEQ ID NO:19, position 624 of SEQ ID NO:20, position 658 of SEQ ID NO:21, position 661 of SEQ ID NO:22, position 661 of SEQ ID NO:23, position 663 of SEQ ID NO:24, position 660 of SEQ ID NO:25, position 649 of SEQ ID NO:26, position 624 of SEQ ID NO:27, or position 691 of SEQ ID NO:28; or position 661 of SEQ ID NO:45 or position 649 of SEQ ID NO:46 and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of a position that contains an adenine at a position corresponding to position 624 set forth in SEQ ID NO:47, position 624 set forth in SEQ ID NO:48, position 658 set forth in SEQ ID NO:49, position 661 set forth in SEQ ID NO:50, position 661 set forth in SEQ ID NO:51, position 663 set forth in SEQ ID NO:52, position 660 set forth in SEQ ID NO:53, position 649 set forth in SEQ ID NO:54, position 624 set forth in SEQ ID NO:55, or position 691 set forth in SEQ ID NO:56;

[0091] Similar amplicons can be generated from mRNA and / or cDNA sequences. PCR primer pairs can be derived from known sequences, for example, using computer programs designed for that purpose, such as the PCR primer analysis tools in Vector NTI 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.). Additionally, sequences can be visually inspected and primers manually specified using known guidelines.

[0092] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including the use of labeled primers or probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may 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 (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0093] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold or more above background, including more than 10-fold above background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0094] Suitable stringent conditions that promote 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 or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. +ion, usually about 0.01 to 1.0 M Na + The conditions will be ionic concentration (or other salts) and temperature of at least about 30° C. for short probes (e.g., 10-50 nucleotides) and at least about 60° C. for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0095] The present disclosure also provides a method for detecting the presence of a predicted CREB3L3 loss-of-function polypeptide, comprising performing an assay on a biological sample obtained from a subject to determine whether the CREB3L3 polypeptide in the biological sample contains one or more mutations that cause the polypeptide to have a loss of function (partial or complete) or a predicted loss of function (partial or complete). The predicted CREB3L3 loss-of-function polypeptide can be any of the predicted CREB3L3 loss-of-function polypeptides described herein. In some embodiments, the method detects the presence of CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, Asp182Asn-D, or Asp181Asn. In some embodiments, the method detects the presence of CREB3L3 Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, or Asp182Asn-D.

[0096] In some embodiments, the method includes performing an assay on a biological sample obtained from the subject to determine whether the CREB3L3 polypeptide in the biological sample contains an asparagine at a position corresponding to position 182 set forth in SEQ ID NO:64, an asparagine at a position corresponding to position 182 set forth in SEQ ID NO:65, an asparagine at a position corresponding to position 182 set forth in SEQ ID NO:66, an asparagine at a position corresponding to position 182 set forth in SEQ ID NO:67, or an asparagine at a position corresponding to position 181 set forth in SEQ ID NO:68.

[0097] In some embodiments, the detection step includes sequencing at least a portion of the CREB3L3 polypeptide including a position corresponding to position 182 set forth in SEQ ID NO:64, position 182 set forth in SEQ ID NO:65, position 182 set forth in SEQ ID NO:66, position 182 set forth in SEQ ID NO:67, position 181 set forth in SEQ ID NO:68, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0098] In some embodiments, the detection step includes an immunoassay to detect the presence of a CREB3L3 polypeptide including a position corresponding to position 182 set forth in SEQ ID NO:64, position 182 set forth in SEQ ID NO:65, position 182 set forth in SEQ ID NO:66, or position 182 set forth in SEQ ID NO:67, or SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63.

[0099] In some embodiments, if the subject does not have CREB3L3 predicted loss-of-function polypeptide, the subject has high risk of developing liver disease, or parenchymal liver disease, liver fibrosis, liver cirrhosis or NAFLD.In some embodiments, if the subject has CREB3L3 predicted loss-of-function polypeptide, the subject has low risk of developing liver disease, or parenchymal liver disease, liver fibrosis, liver cirrhosis or NAFLD.

[0100] The present disclosure also provides isolated nucleic acid molecules that hybridize with a CREB3L3 mutant genomic nucleic acid molecule, a CREB3L3 mutant mRNA molecule, and / or a CREB3L3 mutant cDNA molecule (e.g., any of the genomic mutant nucleic acid molecules, mRNA mutant molecules, and cDNA mutant molecules disclosed herein). In some embodiments, such isolated nucleic acid molecules hybridize with a CREB3L3 mutant nucleic acid molecule under stringent conditions. Such nucleic acid molecules can be used, for example, as probes, primers, mutation-specific probes, or mutation-specific primers as described or exemplified herein.

[0101] In some embodiments, the isolated nucleic acid molecule is selected from the group consisting of position 6,120 set forth in SEQ ID NO:2, position 661 set forth in SEQ ID NO:17, position 649 set forth in SEQ ID NO:18, position 624 set forth in SEQ ID NO:19, position 624 set forth in SEQ ID NO:20, position 658 set forth in SEQ ID NO:21, position 661 set forth in SEQ ID NO:22, position 661 set forth in SEQ ID NO:23, position 663 set forth in SEQ ID NO:24, position 660 set forth in SEQ ID NO:25, position 649 set forth in SEQ ID NO:26, position 624 set forth in SEQ ID NO:27, and position 691 set forth in SEQ ID NO:28. , and hybridizes to a portion of a CREB3L3 nucleic acid molecule including a position corresponding to position 661 set forth in SEQ ID NO:45, position 649 set forth in SEQ ID NO:46, position 624 set forth in SEQ ID NO:47, position 624 set forth in SEQ ID NO:48, position 658 set forth in SEQ ID NO:49, position 661 set forth in SEQ ID NO:50, position 661 set forth in SEQ ID NO:51, position 663 set forth in SEQ ID NO:52, position 660 set forth in SEQ ID NO:53, position 649 set forth in SEQ ID NO:54, position 624 set forth in SEQ ID NO:55, or position 691 set forth in SEQ ID NO:56.

[0102] In some embodiments, such isolated nucleic acid molecules comprise 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, 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, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise 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, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists 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 some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0103] In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 contiguous nucleotides of a nucleic acid molecule that is 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% identical to a CREB3L3 variant genomic nucleic acid molecule, a CREB3L3 variant mRNA molecule, and / or a CREB3L3 variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0104] In some embodiments, the isolated mutation-specific probe or mutation-specific primer comprises at least about 15 nucleotides, wherein the mutation-specific probe or mutation-specific primer comprises a nucleotide sequence that is complementary to a nucleotide sequence of a portion of a CREB3L3 nucleic acid molecule or its complement that encodes a loss-of-function polypeptide predicted in CREB3L3. In some embodiments, the portion is selected from the group consisting of position 6,120 set forth in SEQ ID NO:2, or its complement; position 661 set forth in SEQ ID NO:17, or its complement; position 649 set forth in SEQ ID NO:18, or its complement; position 624 set forth in SEQ ID NO:19, or its complement; position 624 set forth in SEQ ID NO:20, or its complement; position 658 set forth in SEQ ID NO:21, or its complement; position 661 set forth in SEQ ID NO:22, or its complement; position 661 set forth in SEQ ID NO:23, or its complement; position 663 set forth in SEQ ID NO:24, or its complement; position 660 set forth in SEQ ID NO:25, or its complement; position 649 set forth in SEQ ID NO:26, or its complement; position 624 set forth in SEQ ID NO:27, or its complement; position 658 set forth in SEQ ID NO:21, or its complement; position 691, or its complement; position 661 set forth in SEQ ID NO: 45, or its complement; position 649 set forth in SEQ ID NO: 46, or its complement; position 624 set forth in SEQ ID NO: 47, or its complement; position 624 set forth in SEQ ID NO: 48, or its complement; position 658 set forth in SEQ ID NO: 49, or its complement; position 661 set forth in SEQ ID NO: 50, or its complement; position 661 set forth in SEQ ID NO: 51, or its complement; position 663 set forth in SEQ ID NO: 52, or its complement; position 660 set forth in SEQ ID NO: 53, or its complement; position 649 set forth in SEQ ID NO: 54, or its complement; position 624 set forth in SEQ ID NO: 55, or its complement; or position 691 set forth in SEQ ID NO: 56, or its complement.In some embodiments, the portion is selected from the group consisting of positions 6,120 to 6,122 set forth in SEQ ID NO:2, or a complement thereof; positions 661 to 663 set forth in SEQ ID NO:17, or a complement thereof; positions 649 to 651 set forth in SEQ ID NO:18, or a complement thereof; positions 624 to 626 set forth in SEQ ID NO:19, or a complement thereof; positions 624 to 626 set forth in SEQ ID NO:20, or a complement thereof; positions 658 to 660 set forth in SEQ ID NO:21, or a complement thereof; positions 661 to 663 set forth in SEQ ID NO:22, or a complement thereof; positions 661 to 663 set forth in SEQ ID NO:23, or a complement thereof; positions 663 to 665 set forth in SEQ ID NO:24, or a complement thereof; positions 660 to 662 set forth in SEQ ID NO:25, or a complement thereof; positions 649 to 651 set forth in SEQ ID NO:26, or a complement thereof; positions 624 to 626 set forth in SEQ ID NO:27, or a complement thereof; positions 625 to 626 set forth in SEQ ID NO:28, or a complement thereof. positions 91 to 693, or a complement thereof; positions 661 to 663 in SEQ ID NO: 45, or a complement thereof; positions 649 to 651 in SEQ ID NO: 46, or a complement thereof; positions 624 to 626 in SEQ ID NO: 47, or a complement thereof; positions 624 to 626 in SEQ ID NO: 48, or a complement thereof; positions 658 to 660 in SEQ ID NO: 49, or a complement thereof; positions 661 to 663 in SEQ ID NO: 50, or a complement thereof Complement; positions 661 to 663 set forth in SEQ ID NO: 51, or their complement; positions 663 to 665 set forth in SEQ ID NO: 52, or their complement; positions 660 to 662 set forth in SEQ ID NO: 53, or their complement; positions 649 to 651 set forth in SEQ ID NO: 54, or their complement; positions 624 to 626 set forth in SEQ ID NO: 55, or their complement; or positions 691 to 693 set forth in SEQ ID NO: 56, or their complement.

[0105] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0106] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0107] In some embodiments, the primers can be used in second generation or high throughput sequencing, including mutation specific primers. Sometimes the primers can be modified, including mutation specific primers. In particular, the primers can include various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is typically performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading and emulsion PCR steps. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain 5'-biotin tags for immobilizing the DNA library on streptavidin-coated beads.

[0108] The probes and primers described herein can be used to detect nucleotide variations within any of the CREB3L3 mutant genomic nucleic acid molecules, CREB3L3 mutant mRNA molecules, and / or CREB3L3 mutant cDNA molecules disclosed herein. The primers described herein can be used to amplify CREB3L3 mutant genomic nucleic acid molecules, CREB3L3 mutant mRNA molecules, or CREB3L3 mutant cDNA molecules, or fragments thereof.

[0109] The present disclosure also provides a pair of primers comprising any of the primers described above. For example, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 6,120 of SEQ ID NO:1 in a particular CREB3L3 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a CREB3L3-based genomic nucleic acid molecule. Conversely, if one of the 3' ends of the primer hybridizes with adenine (rather than guanine) at a position corresponding to position 6,120 of SEQ ID NO:2 in a particular CREB3L3 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a CREB3L3 variant genomic nucleic acid molecule. In some embodiments, the nucleotide of the primer that is complementary to the adenine at a position corresponding to position 6,120 of SEQ ID NO:2 can be at the 3' end of the primer. In addition, if one of the 3' ends of the primer hybridizes with guanine (rather than adenine) at a position corresponding to position 661 of SEQ ID NO:3 in a particular CREB3L3 nucleic acid molecule, the presence of the amplified fragment indicates the presence of a CREB3L3-based mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) in a particular CREB3L3 mRNA molecule at a position corresponding to position 661 set forth in SEQ ID NO: 17, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at the position corresponding to position 661 set forth in SEQ ID NO: 17 can be at the 3' end of the primer.

[0110] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 649 of SEQ ID NO: 4 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 649 of SEQ ID NO: 18 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 649 of SEQ ID NO: 18 can be at the 3' end of the primer.

[0111] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 624 of SEQ ID NO:5 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 624 of SEQ ID NO:19 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 624 of SEQ ID NO:19 can be at the 3' end of the primer.

[0112] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 624 of SEQ ID NO:6 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 624 of SEQ ID NO:20 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 624 of SEQ ID NO:20 can be at the 3' end of the primer.

[0113] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 658 of SEQ ID NO:7 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 658 of SEQ ID NO:21 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 658 of SEQ ID NO:21 can be at the 3' end of the primer.

[0114] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 661 of SEQ ID NO:8 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 661 of SEQ ID NO:22 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 661 of SEQ ID NO:22 can be at the 3' end of the primer.

[0115] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 661 of SEQ ID NO:9 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 661 of SEQ ID NO:23 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 661 of SEQ ID NO:23 can be at the 3' end of the primer.

[0116] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 663 of SEQ ID NO: 10 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 663 of SEQ ID NO: 24 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 663 of SEQ ID NO: 24 can be at the 3' end of the primer.

[0117] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 663 of SEQ ID NO: 11 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 660 of SEQ ID NO: 25 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 660 of SEQ ID NO: 25 can be at the 3' end of the primer.

[0118] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 649 of SEQ ID NO: 12 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 649 of SEQ ID NO: 26 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 649 of SEQ ID NO: 26 can be at the 3' end of the primer.

[0119] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 624 of SEQ ID NO: 13 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 624 of SEQ ID NO: 27 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 624 of SEQ ID NO: 27 can be at the 3' end of the primer.

[0120] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 691 of SEQ ID NO: 14 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference mRNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 691 of SEQ ID NO: 28 in a particular CREB3L3 mRNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 mutant mRNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 691 of SEQ ID NO: 28 can be at the 3' end of the primer.

[0121] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 661 of SEQ ID NO: 31 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 661 of SEQ ID NO: 45 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 661 of SEQ ID NO: 45 can be at the 3' end of the primer.

[0122] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 649 of SEQ ID NO: 32 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 649 of SEQ ID NO: 46 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at the position corresponding to position 649 of SEQ ID NO: 46 can be at the 3' end of the primer.

[0123] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 624 of SEQ ID NO: 33 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 624 of SEQ ID NO: 47 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 624 of SEQ ID NO: 47 can be at the 3' end of the primer.

[0124] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 624 of SEQ ID NO: 31 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 624 of SEQ ID NO: 48 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 624 of SEQ ID NO: 48 can be at the 3' end of the primer.

[0125] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 658 of SEQ ID NO: 35 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 658 of SEQ ID NO: 49 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 658 of SEQ ID NO: 49 can be at the 3' end of the primer.

[0126] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 661 of SEQ ID NO: 36 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 661 of SEQ ID NO: 50 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 661 of SEQ ID NO: 50 can be at the 3' end of the primer.

[0127] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 661 of SEQ ID NO: 37 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 661 of SEQ ID NO: 51 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 661 of SEQ ID NO: 51 can be at the 3' end of the primer.

[0128] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 663 of SEQ ID NO: 38 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 663 of SEQ ID NO: 52 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at the position corresponding to position 663 of SEQ ID NO: 52 can be at the 3' end of the primer.

[0129] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 663 of SEQ ID NO: 39 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 660 of SEQ ID NO: 53 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at the position corresponding to position 660 of SEQ ID NO: 53 can be at the 3' end of the primer.

[0130] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 649 of SEQ ID NO: 40 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 649 of SEQ ID NO: 54 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at the position corresponding to position 649 of SEQ ID NO: 54 can be at the 3' end of the primer.

[0131] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 624 of SEQ ID NO: 41 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 624 of SEQ ID NO: 55 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 624 of SEQ ID NO: 55 can be at the 3' end of the primer.

[0132] If one of the 3' ends of the primer hybridizes to a guanine (rather than an adenine) at a position corresponding to position 691 of SEQ ID NO: 42 in a particular CREB3L3 nucleic acid molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 reference cDNA molecule. Conversely, if one of the 3' ends of the primer hybridizes to an adenine (rather than a guanine) at a position corresponding to position 691 of SEQ ID NO: 56 in a particular CREB3L3 cDNA molecule, the presence of an amplified fragment indicates the presence of a CREB3L3 variant cDNA molecule. In some embodiments, the nucleotide of the primer complementary to the adenine at a position corresponding to position 691 of SEQ ID NO: 56 can be at the 3' end of the primer.

[0133] In the context of the present disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleic acid sequence encoding a CREB3L3-based genomic nucleic acid molecule, a CREB3L3-based mRNA molecule, and / or a CREB3L3-based cDNA molecule.

[0134] In any of the embodiments described throughout this disclosure, the probe (e.g., the mutation-specific probe) can include a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0135] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which a molecule, such as any of the probes disclosed herein, can associate. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which a plurality of different probes are attached in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish, such as a standard 96-well format. In some embodiments, a multi-well glass slide can be used, usually containing one array per well. In some embodiments, the support is a microarray.

[0136] In some embodiments, any of the methods described herein may further include determining the gene burden of the subject having a CREB3L3 variant nucleic acid molecule (e.g., a CREB3L3 variant nucleic acid molecule (e.g., a CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in CREB3L3, etc.), and / or a predicted loss-of-function variant polypeptide in CREB3L3 associated with a reduced risk of developing liver disease. The gene burden is the sum of all variants in the CREB3L3 gene, which may be performed in an association analysis with liver disease. In some embodiments, the subject is homozygous for one or more of the CREB3L3 variant nucleic acid molecules associated with a reduced risk of developing liver disease. In some embodiments, the subject is heterozygous for one or more of the CREB3L3 variant nucleic acid molecules associated with a reduced risk of developing liver disease. The results of the association analysis may include determining the gene burden of the subject having a CREB3L3 variant nucleic acid molecule (e.g., a CREB3L3 variant nucleic acid molecule (e.g., a CREB3L3 variant nucleic acid molecule encoding a predicted loss-of-function variant polypeptide in CREB3L3, etc.), and / or a predicted loss-of-function variant polypeptide in CREB3L3 associated with a reduced risk of developing liver disease. This suggests that CREB3L3 variant nucleic acid molecules (e.g., CREB3L3 variant nucleic acid molecules encoding a CREB3L3 polypeptide) are associated with a reduced risk of developing liver disease. If a subject has a lower gene burden, the subject is at a higher risk of developing liver disease, and the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating, preventing, or inhibiting liver disease and / or a CREB3L3 inhibitor. If a subject has a higher gene burden, the subject is at a lower risk of developing liver disease, and the subject is administered or continues to be administered a standard or lower dose of a therapeutic agent for treating, preventing, or inhibiting liver disease. The higher the gene burden, the lower the risk of developing liver disease. Table 2 lists representative CREB3L3 variant nucleic acid molecules that can be used for gene burden analysis. The association between CREB3L3 and liver phenotypes was induced by multiple rare pLOF variants in the gene. In some embodiments, the gene burden analysis includes the variant 19:4159750:G:A (Asp182Asn).

[0137] Table 2: Predicted loss-of-function variants with alternative allele frequencies <1% in CREB3L3 identified by exome sequencing and included in gene burden association analyses

[0138] [Table 2-1]

[0139] [Table 2-2]

[0140] [Table 2-3]

[0141] [Table 2-4]

[0142] [Table 2-5]

[0143] [Table 2-6]

[0144] [Table 2-7]

[0145] In some embodiments, the genetic burden of a subject having any one or more CREB3L3 variant nucleic acid molecules (e.g., a CREB3L3 variant nucleic acid molecule (e.g., a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted for CREB3L3) represents a weighted sum of any multiple of the CREB3L3 variant nucleic acid molecules. In some embodiments, the genetic burden represents a weighted sum of at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, 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 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 23 The gene burden is calculated using at least about 1,000, at least about 10,000, at least about 100,000, or at least about 1,000,000 or more gene variants, where the gene burden is the number of alleles multiplied by the association estimate with liver disease or related outcomes for each allele (e.g., weighted polygenic burden score). This can include any gene variants that are close to the CREB3L3 gene (up to 10Mb around the gene) that show non-zero association with liver disease-related traits in gene association analysis, regardless of genome annotation. In some embodiments, if a subject has a gene burden higher than the desired threshold score, the subject has a low risk of developing liver disease. In some embodiments, if a subject has a gene burden lower than the desired threshold score, the subject has a high risk of developing liver disease.

[0146] In some embodiments, the gene burden can be divided into quintiles, such as upper quintile, middle quintile, and lower quintile, with the upper quintile of gene burden corresponding to the lowest risk group, and the lower quintile of gene burden corresponding to the highest risk group.In some embodiments, the subject with the larger gene burden comprises the highest weighted gene burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% gene burden from the subject population.In some embodiments, the gene variant comprises a gene variant that has an association with liver disease in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range for the association. In some embodiments, each of the identified genetic variants includes a genetic variant that has an association with liver disease with a p-value of about 10-2, about 10-3, about 10-4, about 10-5, about 10-6, about 10-7, about 10-8, about 10-9, about 10-10, about 10-11, about 10-12, about 10-13, about 10-14, or about 10-15 or less. In some embodiments, the identified genetic variants include a genetic variant that has an association with liver disease with a p-value of less than 5×10-8. In some embodiments, the identified genetic variants include genetic variants that have an association with liver disease in subjects at high risk compared to the remainder of a reference population where the odds ratio (OR) is about 1.5 or more, about 1.75 or more, about 2.0 or more, or about 2.25 or more for the top 20% of the distribution; or about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or may be greater than 7.0. In some embodiments, high-risk subjects include subjects with a genetic burden in the bottom decile, quintile, or tertile in the reference population. The genetic burden threshold is determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0147] In some embodiments, once a subject is identified as having a high risk of developing liver disease, the subject is further administered a therapeutic agent and / or a CREB3L3 inhibitor that treats, prevents, or inhibits liver disease, as described herein. For example, if a subject has a CREB3L3 baseline and is therefore at high risk of developing liver disease, the subject is administered a CREB3L3 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent that treats, prevents, or inhibits liver disease. In some embodiments, if the subject is heterozygous for a CREB3L3 mutant nucleic acid molecule (e.g., a CREB3L3 mutant nucleic acid molecule (e.g., a CREB3L3 mutant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3), the subject is administered a therapeutic agent that treats, prevents, or inhibits liver disease at a dosage that is the same as or less than a standard dosage, and is also administered a CREB3L3 inhibitor. In some embodiments, the subject is CREB3L3 norm. In some embodiments, the subject is heterozygous for a CREB3L3 mutant nucleic acid molecule. Further, if the subject has a CREB3L3 mutant nucleic acid molecule (e.g., a CREB3L3 mutant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3), If a subject has a lower genetic burden for CREB3L3 variant nucleic acid molecule (such as CREB3L3 variant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3), the subject is administered a therapeutic agent for treating, preventing or inhibiting liver disease at a dose equal to or higher than the standard dose administered to a subject with a higher genetic burden for CREB3L3 variant nucleic acid molecule (such as CREB3L3 variant nucleic acid molecule that encodes a loss-of-function polypeptide predicted by CREB3L3).

[0148] The nucleotide sequence of the CREB3L3 reference genomic nucleic acid molecule is set forth in SEQ ID NO: 1. With reference to SEQ ID NO: 1, position 6,120 is a guanine. A CREB3L3 mutant genomic nucleic acid molecule exists in which the guanine at position 6,120 is replaced with an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:2.

[0149] The nucleotide sequence of the CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 3. With reference to SEQ ID NO: 3, position 661 is a guanine. The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 4. With reference to SEQ ID NO: 4, position 649 is a guanine.

[0150] The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 5. With reference to SEQ ID NO: 5, position 624 is a guanine. The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 6. With reference to SEQ ID NO: 6, position 624 is a guanine.

[0151] The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 7. With reference to SEQ ID NO: 7, position 658 is a guanine. The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 8. With reference to SEQ ID NO: 8, position 661 is a guanine.

[0152] The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 9. With reference to SEQ ID NO: 9, position 661 is a guanine. The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 10. With reference to SEQ ID NO: 10, position 663 is a guanine.

[0153] The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 11. With reference to SEQ ID NO: 11, position 663 is a guanine. The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 12. With reference to SEQ ID NO: 12, position 649 is a guanine.

[0154] The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 13. With reference to SEQ ID NO: 13, position 624 is a guanine. The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 14. With reference to SEQ ID NO: 14, position 691 is a guanine.

[0155] The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 15. With reference to SEQ ID NO: 15, position 660 is a guanine. The nucleotide sequence of another CREB3L3 reference mRNA molecule is set forth in SEQ ID NO: 16. With reference to SEQ ID NO: 16, position 620 is a guanine.

[0156] A CREB3L3 mutant mRNA molecule exists in which the guanine at position 661 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:17.

[0157] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 649 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:18.

[0158] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 624 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:19.

[0159] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 624 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:20.

[0160] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 658 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:21.

[0161] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 661 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:22.

[0162] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 661 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:23.

[0163] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 663 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:24.

[0164] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 663 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:25.

[0165] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 649 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:26.

[0166] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 624 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:27.

[0167] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 691 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:28.

[0168] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 660 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:29.

[0169] Another CREB3L3 mutant mRNA molecule exists in which the guanine at position 620 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:30.

[0170] The nucleotide sequence of the CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 31. With reference to SEQ ID NO: 31, position 661 is a guanine. The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 32. With reference to SEQ ID NO: 32, position 649 is a guanine.

[0171] The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 33. With reference to SEQ ID NO: 33, position 624 is a guanine. The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 34. With reference to SEQ ID NO: 34, position 624 is a guanine.

[0172] The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 35. With reference to SEQ ID NO: 35, position 658 is a guanine. The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 36. With reference to SEQ ID NO: 36, position 661 is a guanine.

[0173] The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 37. With reference to SEQ ID NO: 37, position 661 is a guanine. The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 38. With reference to SEQ ID NO: 38, position 663 is a guanine.

[0174] The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 39. With reference to SEQ ID NO: 39, position 663 is a guanine. The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 40. With reference to SEQ ID NO: 40, position 649 is a guanine.

[0175] The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 41. With reference to SEQ ID NO: 41, position 624 is a guanine. The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 42. With reference to SEQ ID NO: 42, position 691 is a guanine.

[0176] The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 43. With reference to SEQ ID NO: 43, position 660 is a guanine. The nucleotide sequence of another CREB3L3 reference cDNA molecule is set forth in SEQ ID NO: 43. With reference to SEQ ID NO: 43, position 620 is a guanine.

[0177] A CREB3L3 mutant cDNA molecule exists in which the guanine at position 661 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:45.

[0178] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 649 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:46.

[0179] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 624 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:47.

[0180] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 624 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:48.

[0181] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 658 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:49.

[0182] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 661 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:50.

[0183] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 661 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:51.

[0184] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 663 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:52.

[0185] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 663 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:53.

[0186] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 649 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:54.

[0187] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 624 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:55.

[0188] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 691 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:56.

[0189] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 660 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:57.

[0190] Another CREB3L3 mutant cDNA molecule exists in which the guanine at position 620 is replaced by an adenine, the nucleotide sequence of which is set forth in SEQ ID NO:58.

[0191] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be derived from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be an ortholog from human or another organism (e.g., non-human mammal, rodent, mouse, or rat). It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are only exemplary sequences. Other sequences are also possible.

[0192] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, and external guide sequences. Functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecules.

[0193] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, for example in a vector, or heterologous labels. For example, the isolated nucleic acid molecules disclosed herein can be present as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent materials; metal-containing materials; or enzymes, where enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate to detect the presence of HRP. Exemplary labels 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 tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0194] The isolated nucleic acid molecule or its complement can also be present in a host cell. In some embodiments, the host cell can comprise a vector comprising any of the nucleic acid molecules described herein or their complements. In some embodiments, the nucleic acid molecule is operably linked to a promoter active in the host cell. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell.

[0195] The disclosed nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0196] The nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of 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-aminoadenine-9-yl. Modified bases include 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-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, cytosine and thymine, 5- Examples include, but are not limited to, uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0197] Nucleotide analogs can also include modifications of 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C2~10 Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1~10 Examples of suitable substituents include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may be made at other positions on the sugar, particularly the 3' position of the sugar in the 3' terminal nucleotide or 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0198] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0199] The present disclosure also provides a vector comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a 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. 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 (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0200] Desirable regulatory sequences for mammalian host cell expression may include, for example, viral elements directing high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells, such as 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 (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0201] Percent identity (or percent complementarity) between specific stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using 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 the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that utilize the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percent sequence identity is preferred over a lower one.

[0202] The present disclosure also provides a composition comprising one or more of the isolated nucleic acid molecules, genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. 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, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, and the like.

[0203] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of the specified reference sequence when the particular nucleotide or sequence of nucleotides is compared to a reference sequence (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:31). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to the reference sequence, not by the actual position number of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of the residues in the particular nucleotide or nucleotide sequence is done with reference to the reference sequence to which it is aligned.

[0204] For example, a CREB3L3 nucleic acid molecule comprising a nucleotide sequence encoding a loss-of-function polypeptide predicted by CREB3L3, the nucleotide sequence of which contains an adenine at position corresponding to position 6,120 of SEQ ID NO: 2, means that when the nucleotide sequence of the CREB3L3 genomic nucleic acid molecule is aligned against the sequence of SEQ ID NO: 2, the sequence of CREB3L3 has an adenine residue at position corresponding to position 6,120 of SEQ ID NO: 2. The same applies to a CREB3L3 mRNA molecule comprising a nucleotide sequence encoding a loss-of-function polypeptide predicted by CREB3L3, the nucleotide sequence of which contains an adenine at position corresponding to position 661 of SEQ ID NO: 17, and to a CREB3L3 cDNA molecule comprising a nucleotide sequence encoding a loss-of-function polypeptide predicted by CREB3L3, the nucleotide sequence of which contains an adenine at position corresponding to position 661 of SEQ ID NO: 45. In other words, these terms refer to a nucleic acid molecule encoding a CREB3L3 polypeptide, where a genomic nucleic acid molecule has a nucleotide sequence that includes an adenine residue that is homologous to the adenine residue at position 6,120 of SEQ ID NO:2 (or an mRNA molecule has a nucleotide sequence that includes an adenine residue that is homologous to the adenine residue at position 661 of SEQ ID NO:17, or a cDNA molecule has a nucleotide sequence that includes an adenine residue that is homologous to the adenine residue at position 661 of SEQ ID NO:45).

[0205] As described herein, the position in the CREB3L3 genomic nucleic acid molecule corresponding to position 6,120 of SEQ ID NO:2 can be identified, for example, by performing sequence comparison between the nucleotide sequence of a particular CREB3L3 nucleic acid molecule and the nucleotide sequence of SEQ ID NO:2. For example, there are various computer algorithms that can be used to perform sequence comparison to identify the position of the nucleotide corresponding to position 6,120 of SEQ ID NO:2. For example, sequence comparison can be performed by using the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res., 1997, 25, 3389-3402) or CLUSTALW software (Sievers and Higgins, Methods Mol. Biol., 2014, 1079, 105-116). However, sequences can also be aligned manually.

[0206] The amino acid sequences of the CREB3L3 reference polypeptides are set forth in SEQ ID NO:59 (isoform 1), SEQ ID NO:60 (isoform 2), SEQ ID NO:61 (isoform 3), SEQ ID NO:62 (isoform 4), and SEQ ID NO:63 (isoform 5).

[0207] With reference to SEQ ID NO:59 (isoform 1), the CREB3L3 reference polypeptide is 467 amino acids in length. With reference to SEQ ID NO:59, position 182 is an aspartic acid. With reference to SEQ ID NO:60 (isoform 2), the CREB3L3 reference polypeptide is 459 amino acids in length. With reference to SEQ ID NO:60, position 182 is an aspartic acid.

[0208] With reference to SEQ ID NO:61 (isoform 3), the CREB3L3 reference polypeptide is 337 amino acids in length. With reference to SEQ ID NO:61, position 182 is an aspartic acid. With reference to SEQ ID NO:62 (isoform 4), the CREB3L3 reference polypeptide is 473 amino acids in length. With reference to SEQ ID NO:62, position 182 is an aspartic acid.

[0209] With reference to SEQ ID NO:63 (isoform 5), the CREB3L3 reference polypeptide is 473 amino acids in length. With reference to SEQ ID NO:63, position 181 is an aspartic acid. The amino acid sequences of the loss-of-function polypeptides predicted by CREB3L3 are set forth in SEQ ID NO:64 (isoform 1), SEQ ID NO:65 (isoform 2), SEQ ID NO:66 (isoform 3), SEQ ID NO:67 (isoform 4), and SEQ ID NO:68 (isoform 5). With reference to SEQ ID NO:64 (Asp182Asn-A; isoform 1), position 182 is asparagine. With reference to SEQ ID NO:65 (Asp182Asn-B; isoform 2), position 182 is asparagine. With reference to SEQ ID NO:66 (Asp182Asn-C; isoform 3), position 182 is asparagine. With reference to SEQ ID NO:67 (Asp182Asn-D; isoform 4), position 182 is asparagine. With reference to SEQ ID NO:68 (Asp181Asn; isoform 5), position 181 is asparagine.

[0210] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding toward the 3'-end (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to the shown strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each sequence).

[0211] The present disclosure also provides a therapeutic agent for treating or inhibiting liver disease for use in treating liver disease in a subject (or for use in preparing a medicament for treating liver disease), wherein the subject has any of the CREB3L3 mutant genomic nucleic acid molecules, mutant mRNA molecules, and / or mutant cDNA molecules encoding a predicted loss-of-function polypeptide of CREB3L3 described herein. The therapeutic agent for treating or inhibiting liver disease can be any of the therapeutic agents for treating or inhibiting liver disease described herein.

[0212] In some embodiments, the subject is identified as having a genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted for CREB3L3 having a nucleotide sequence including an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, or its complement.

[0213] In some embodiments, the subject has an adenine at a position corresponding to 661 in SEQ ID NO: 17, or its complement; an adenine at a position corresponding to 649 in SEQ ID NO: 18, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO: 19, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO: 20, or its complement; an adenine at a position corresponding to 658 in SEQ ID NO: 21, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO: 22, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO: 23, or its complement. The CREB3L3-targeted mRNA molecule is identified as having an mRNA molecule encoding a loss-of-function polypeptide predicted for CREB3L3, the nucleotide sequence of which includes an adenine, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:28, or its complement.

[0214] In some embodiments, the subject has an adenine at a position corresponding to 661 set forth in SEQ ID NO:45, or its complement; an adenine at a position corresponding to 649 set forth in SEQ ID NO:46, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:47, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:48, or its complement; an adenine at a position corresponding to 658 set forth in SEQ ID NO:49, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:50, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:51, or its complement. The present invention is identified as having a cDNA molecule encoding a loss-of-function polypeptide predicted for CREB3L3, the cDNA molecule having a nucleotide sequence including: adenine, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or its complement.

[0215] In some embodiments, the subject is provided with a genomic nucleic acid molecule having i) a nucleotide sequence encoding a CREB3L3 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an adenine at a position corresponding to position 6,120 set forth in SEQ ID NO:2, or a complement thereof; ii) a genomic nucleic acid molecule having a nucleotide sequence encoding a CREB3L3 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an adenine at a position corresponding to position 661 set forth in SEQ ID NO:17, or a complement thereof; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:18, or a complement thereof; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:19, or a complement thereof; an adenine at a position corresponding to position 624 in SEQ ID NO:0, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:26 or its complement; an adenine at position 624 as set forth in SEQ ID NO:27, or its complement; or an adenine at position 691 as set forth in SEQ ID NO:28, or its complement; or iii) an mRNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of CREB3L3, the nucleotide sequence of which comprises an adenine at position 661 as set forth in SEQ ID NO:45, or its complement; an adenine at position 649 as set forth in SEQ ID NO:46, or its complement; an adenine at position 624 as set forth in SEQ ID NO:47, or its complement; an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:48; an adenine, or its complement, at a position corresponding to position 658 in SEQ ID NO:49; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:50; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:51; an adenine, or its complement, at a position corresponding to position 663 in SEQ ID NO:52; an adenine, or its complement, at a position corresponding to position 660 in SEQ ID NO:53;The subject is identified as having a cDNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of CREB3L3, the nucleotide sequence including an adenine at a position corresponding to position 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or its complement;

[0216] In some embodiments, the subject is identified as having a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted for CREB3L3, wherein the nucleotide sequence includes an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, or its complement.

[0217] In some embodiments, the subject has a nucleotide sequence comprising an adenine at a position corresponding to 661 in SEQ ID NO:17, or its complement; an adenine at a position corresponding to 649 in SEQ ID NO:18, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:19, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO:23, or its complement; an adenine, or its complement, at a position corresponding to position 663 set forth in SEQ ID NO:24; an adenine, or its complement, at a position corresponding to position 660 set forth in SEQ ID NO:25; an adenine, or its complement, at a position corresponding to position 649 set forth in SEQ ID NO:26; an adenine, or its complement, at a position corresponding to position 624 set forth in SEQ ID NO:27; or an adenine, or its complement, at a position corresponding to position 691 set forth in SEQ ID NO:28.

[0218] In some embodiments, the subject has a nucleotide sequence comprising: an adenine at a position corresponding to 661 set forth in SEQ ID NO:45, or its complement; an adenine at a position corresponding to 649 set forth in SEQ ID NO:46, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:47, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:48, or its complement; an adenine at a position corresponding to 658 set forth in SEQ ID NO:49, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:50, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:51, or its complement; or adenine or its complement at a position corresponding to position 663 set forth in SEQ ID NO:52; adenine or its complement at a position corresponding to position 660 set forth in SEQ ID NO:53; adenine or its complement at a position corresponding to position 649 set forth in SEQ ID NO:54; adenine or its complement at a position corresponding to position 624 set forth in SEQ ID NO:55; or adenine or its complement at a position corresponding to position 691 set forth in SEQ ID NO:56.

[0219] In some embodiments, the subject is identified as having a predicted loss-of-function polypeptide of CREB3L3 that includes an asparagine at a position corresponding to 182 set forth in SEQ ID NO:64, 182 set forth in SEQ ID NO:65, 182 set forth in SEQ ID NO:66, 182 set forth in SEQ ID NO:67, or 181 set forth in SEQ ID NO:68.

[0220] The present disclosure also provides a CREB3L3 inhibitor for use in treating liver disease (or for use in preparing a medicament for treating liver disease) in a subject, wherein the subject is heterozygous for any of the CREB3L3 mutant genomic nucleic acid molecules, mutant mRNA molecules, and / or mutant cDNA molecules encoding a predicted loss-of-function polypeptide of CREB3L3 described herein, or the subject is a reference for a CREB3L3 genomic nucleic acid molecule, mRNA molecule, or cDNA molecule. The CREB3L3 inhibitor can be any of the CREB3L3 inhibitors described herein.

[0221] In some embodiments, the subject is a reference for a CREB3L3 genomic nucleic acid molecule, a CREB3L3 mRNA molecule, or a CREB3L3 cDNA molecule. In some embodiments, the subject is a reference for a CREB3L3 genomic nucleic acid molecule. In some embodiments, the subject is a reference for a CREB3L3 mRNA molecule. In some embodiments, the subject is a reference for a CREB3L3 cDNA molecule.

[0222] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted for CREB3L3 having a nucleotide sequence including an adenine at position corresponding to position 6,120 of SEQ ID NO:2, or its complement.

[0223] In some embodiments, the subject has an adenine at a position corresponding to 661 in SEQ ID NO:17, or its complement; an adenine at a position corresponding to 649 in SEQ ID NO:18, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:19, or its complement; an adenine at a position corresponding to 624 in SEQ ID NO:20, or its complement; an adenine at a position corresponding to 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to 661 in SEQ ID NO:23, or its complement. or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:28, or its complement.

[0224] In some embodiments, the subject has an adenine at a position corresponding to 661 set forth in SEQ ID NO:45, or its complement; an adenine at a position corresponding to 649 set forth in SEQ ID NO:46, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:47, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:48, or its complement; an adenine at a position corresponding to 658 set forth in SEQ ID NO:49, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:50, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:51, or its complement. or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or its complement.

[0225] In some embodiments, the subject is provided with a genomic nucleic acid molecule having i) a nucleotide sequence encoding a CREB3L3 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an adenine at a position corresponding to position 6,120 set forth in SEQ ID NO:2, or a complement thereof; ii) a genomic nucleic acid molecule having a nucleotide sequence encoding a CREB3L3 predicted loss-of-function polypeptide, wherein the nucleotide sequence comprises an adenine at a position corresponding to position 661 set forth in SEQ ID NO:17, or a complement thereof; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:18, or a complement thereof; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:19, or a complement thereof; an adenine at a position corresponding to position 624 in SEQ ID NO:0, or its complement; an adenine at a position corresponding to position 658 in SEQ ID NO:21, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:22, or its complement; an adenine at a position corresponding to position 661 in SEQ ID NO:23, or its complement; an adenine at a position corresponding to position 663 in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 in SEQ ID NO:26 or its complement; an adenine at position 624 as set forth in SEQ ID NO:27, or its complement; or an adenine at position 691 as set forth in SEQ ID NO:28, or its complement; or iii) an mRNA molecule having a nucleotide sequence encoding a predicted loss-of-function polypeptide of CREB3L3, the nucleotide sequence of which comprises an adenine at position 661 as set forth in SEQ ID NO:45, or its complement; an adenine at position 649 as set forth in SEQ ID NO:46, or its complement; an adenine at position 624 as set forth in SEQ ID NO:47, or its complement; an adenine, or its complement, at a position corresponding to position 624 in SEQ ID NO:48; an adenine, or its complement, at a position corresponding to position 658 in SEQ ID NO:49; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:50; an adenine, or its complement, at a position corresponding to position 661 in SEQ ID NO:51; an adenine, or its complement, at a position corresponding to position 663 in SEQ ID NO:52; an adenine, or its complement, at a position corresponding to position 660 in SEQ ID NO:53;The subject is identified as heterozygous for a cDNA molecule having a nucleotide sequence encoding a CREB3L3 predicted loss-of-function polypeptide comprising an adenine at a position corresponding to 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to 691 set forth in SEQ ID NO:56, or its complement;

[0226] In some embodiments, the subject is identified as heterozygous for a genomic nucleic acid molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted for CREB3L3, the nucleotide sequence comprising an adenine at a position corresponding to position 6,120 of SEQ ID NO:2, or its complement.

[0227] In some embodiments, the subject has a nucleotide sequence comprising an adenine at a position corresponding to 661 set forth in SEQ ID NO:17, or its complement; an adenine at a position corresponding to 649 set forth in SEQ ID NO:18, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:19, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:20, or its complement; an adenine at a position corresponding to 658 set forth in SEQ ID NO:21, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:22, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:23, or its complement. The CREB3L3 polypeptide is identified as being heterozygous for an mRNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted for CREB3L3, the nucleotide sequence including: adenine, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:24, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:25, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:26, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:27, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:28, or its complement.

[0228] In some embodiments, the subject is diagnosed with a nucleotide sequence comprising an adenine at a position corresponding to 661 set forth in SEQ ID NO:45, or its complement; an adenine at a position corresponding to 649 set forth in SEQ ID NO:46, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:47, or its complement; an adenine at a position corresponding to 624 set forth in SEQ ID NO:48, or its complement; an adenine at a position corresponding to 658 set forth in SEQ ID NO:49, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:50, or its complement; an adenine at a position corresponding to 661 set forth in SEQ ID NO:51, or its complement. The CREB3L3 polypeptide is identified as being heterozygous for a cDNA molecule having a nucleotide sequence encoding a loss-of-function polypeptide predicted for CREB3L3, the loss-of-function polypeptide comprising: adenine, or its complement; an adenine at a position corresponding to position 663 set forth in SEQ ID NO:52, or its complement; an adenine at a position corresponding to position 660 set forth in SEQ ID NO:53, or its complement; an adenine at a position corresponding to position 649 set forth in SEQ ID NO:54, or its complement; an adenine at a position corresponding to position 624 set forth in SEQ ID NO:55, or its complement; or an adenine at a position corresponding to position 691 set forth in SEQ ID NO:56, or its complement.

[0229] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual document was specifically and individually indicated to be so incorporated by reference. Where various versions of a sequence are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application to which the accession number refers, if applicable. Similarly, where different versions of publications, websites, etc. have been published at different times, the version last published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure may be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0230] The following examples are provided to further illustrate the embodiments. They are intended to illustrate, not 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 illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure. EXAMPLES

[0231] Example 1: Loss of function of CREB3L3 is associated with reduced liver damage and protection against liver disease as measured by circulating alanine transferase levels in humans To identify genetic factors that contribute to predisposition to or protection against chronic liver disease, exome sequencing was performed in 567,237 participants of European ancestry from the UK Biobank cohort (UKB) and the Geisinger Health System DiscovEHR study (GHS). For each rare genetic missense or pLOF variant in the genome, associations with alanine aminotransferase (ALT), a widely used biomarker of liver damage, were estimated for the burden of rare loss-of-function and missense variants identified by exome sequencing for each gene in the genome. Statistically significant findings were then evaluated for association with clinical diagnosis of chronic liver disease in the UKB, GHS, and SINAI cohorts.

[0232] Whole-exome analysis revealed that the missense genetic variant rs140312652, which encodes an amino acid change in CREB3L3 (Asp182Asn), was found to have a statistically significant exome-wide level (p<5×10 -8 , Table 3) and low ALT (-1.95 U / L, P = 7.9 × 10 -12 ) was identified as being strongly associated with

[0233] Table 3: Asp182Asn in CREB3L3 is associated with reduced liver damage as measured by circulating alanine transferase levels. Results from an inverse variance-weighted meta-analysis in the GHS and UKB cohorts

[0234] [Table 3]

[0235] RR indicates the number of individuals carrying the alternative allele A of rs140312652 in CREB3L3 (19:4159750:G:A, human genome build 38) (homozygous non-carriers); RA indicates the number of individuals carrying a rare missense or pLOF variant in a single CREB3L3 allele (heterozygous carriers); AA indicates the number of individuals carrying a rare missense or pLOF variant in both CREB3L3 alleles (homozygous carriers); AAF indicates the alternative allele frequency of the pLOF or missense allele in CREB3L3 included in the analysis; pLOF indicates predicted loss of function; U / L indicates units per liter; SD indicates standard deviation; CI indicates confidence interval.

[0236] Association of rare (alternative allele frequency <1%) loss-of-function variants in pLOF was also associated with lower ALT (Table 4), indicating that loss of CREB3L3 function is associated with reduced liver damage as measured by ALT.

[0237] Table 4: Rare predicted loss-of-function variants in CREB3L3 are associated with reduced liver damage as measured by circulating alanine transferase levels.

[0238] [Table 4]

[0239] RR indicates the number of individuals not carrying a rare pLOF variant in CREB3L3 (homozygous non-carriers); RA indicates the number of individuals carrying a rare pLOF variant in a single CREB3L3 allele (heterozygous carriers); AA indicates the number of individuals carrying a rare pLOF variant in both CREB3L3 alleles (homozygous carriers); AAF indicates the alternative allele frequency of the pLOF allele in CREB3L3 included in the analysis; pLOF indicates predicted loss of function; U / L indicates units per liter; SD indicates standard deviation; CI indicates confidence interval.

[0240] In this analysis in Table 4, the burden of pLOF CREB3L3 variants with AAF <1% was the exposure variable and ALT levels were the outcome variable. Results are from an inverse variance weighted meta-analysis in the GHS and UKB cohorts. We next estimated the association between Asp182Asn in CREB3L3 and liver disease outcomes. Asp182Asn in CREB3L3 was associated with protection against parenchymal and nonalcoholic liver disease (Table 5). Heterozygous carriers of these genetic variants had 25% lower odds of liver disease compared with noncarriers.

[0241] Table 5: Asp182Asn in CREB3L3 is associated with protection against liver diseases of various etiologies

[0242] [Table 5]

[0243] RR indicates the number of individuals who are homozygous for the reference genotype (homozygote non-carriers); RA indicates the number of individuals who are heterozygous for the gene exposure genotype (heterozygote carriers); AA indicates the number of individuals who are homozygous for the gene exposure genotype (homozygote carriers); AAF indicates the alternative allele frequencies of the gene exposure genotype; CI indicates the confidence interval.

[0244] In this analysis in Table 5, Asp182Asn in CREB3L3 was the exposure variable and liver disease was the outcome variable. Results are from an inverse variance weighted meta-analysis in the GHS, UKB, SINAI, and UPENN-PMBB cohorts. Participation cohort Genetic association studies were conducted in the United Kingdom Biobank (UKB) cohort (Sudlow et al., PLoS Med, 2015, 12, e1001779) and the DiscoverEHR cohort from the Geisinger Health System (GHS) MyCode Community Health Initiative (Carey et al., Genet. Med., 2016, 18, 906-13). UKB is a population-based cohort study of people aged 40-69 years recruited through 22 study centers in the UK in 2006-2010. Over 430,000 participants of European ancestry from UKB with whole-exome sequencing and clinical phenotype data available were included. The Community Health Initiative of the GHS MyCode study is a health system-based cohort of patients from central and eastern Pennsylvania (USA) recruited in 2007-2019. Over 130,000 participants of European ancestry from the GHS with available whole-exome sequencing and clinical phenotype data were included. Associations with liver outcomes included the Mount Sinai BioMe Biobank cohort (SINAI, Cell, 2019, 177, 58-69), The University of Pennsylvania Penn Medicine BioBank (UPENN-PMBB; Park et al., 2020, doi:10.1038 / s41436-019-0625-8).

[0245] Phenotype definition Laboratory values ​​for ALT and other biomarkers were extracted from the electronic health records (EHR) of participants from the GHS. Median values ​​were calculated for all participants with two or more measurements. In the UKB, ALT and other biomarkers were measured by IFCC (International Federation of Clinical Chemistry) assays on a Beckman Coulter AU5800 at the study baseline visit; continuous phenotypic values ​​were transformed by the inverse standard normal function and applied separately to men and women within each ancestry group prior to genetic association analysis.

[0246] Disease outcomes were defined according to the International Classification of Diseases, 10th Revision (ICD-10) and read codes stored in the EHR. Self-reported disease status was used when available. For medical procedures, Office of Population Censuses and Surveys Classification of Interventions and Procedures version 4 (OPCS4) codes were used. Individuals with liver disease were classified using a combination of EHR records, self-reports, and ALT measurements as described in Table 6.

[0247] Table 6: Definitions of liver and coronary disease outcomes in the UKB, GHS, and SINAI cohorts

[0248] [Table 6]

[0249] ICD10 refers to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems; UKB.OPCS4 refers to the Office of Population Census and Surveys (OPCS) Classification of Interventions and Procedures, 4th edition, used in UK Biobank (UKB); UKB.f.20002 refers to self-reported non-cancer disease codes used in UKB; UKB.f.20004 refers to self-reported medical procedures used in UKB.

[0250] Genotype data High-coverage whole-exome sequencing was performed as previously detailed (Science, 2016, 354:aaf6814.; and Nature, 2020, 586:749-756) and summarized below. NimbleGen probes (VCRome, for a portion of the GHS cohort) or a modified version of the xGen design available from Integrated DNA Technologies (IDT, for the remainder of the GHS and other cohorts) were used to capture targeted sequences in the exome. To facilitate multiplexed exome capture and sequencing, a unique 6 base pair (bp) barcode (VCRome) or 10 bp barcode (IDT) was added to each DNA fragment during library preparation. Equal amounts of samples were pooled prior to exome capture. Sequencing was performed on an Illumina v4 HiSeq 2500 instrument (for a portion of the GHS cohort) or on a NovaSeq instrument (for the remainder of the GHS and other cohorts) using 75 bp paired-end reads. Sequencing had sufficient coverage depth (i.e., the number of sequence reads covering each nucleotide in the target region of the genome) to provide >20-fold coverage across 85% of the target bases in 96% of the VCRome samples, and >20-fold coverage across 90% of the target bases in 99% of the IDT samples. Data processing steps included demultiplexing of samples using Illumina software, alignment to the GRCh38 human genome reference sequence including generation of binary alignment and mapping files (BAM), processing of the BAM files (e.g., marking of duplicate reads and other read mapping assessments). Variant calling was performed using the GLNexus system (DOI: 10.1101 / 343970). Variant mapping and annotation was based on the GRCh38 human genome reference sequence and Ensembl v85 gene definitions using snpEff software. snpEff predictions relating to protein-coding transcripts with annotated start and end points were then collapsed into a single functional effect prediction by selecting the most deleterious functional effect class for each gene.The hierarchy of these annotations (from most deleterious to least deleterious) was: frameshift, stop gain, stop loss, splice acceptor, splice donor, stop lost, in-frame indel, missense, and other annotations. Predicted LOF gene variants included a) insertions or deletions resulting in a frameshift, b) insertions, deletions, or single-nucleotide variants resulting in the introduction of a premature stop codon or loss of a transcription start or stop site, and c) donor or acceptor splice site variants. Missense variants were classified for their likely functional impact according to a number of in silico prediction algorithms that predicted deleteriousness using SIFT (Adzhubei et al., Nat. Methods, 2010, 7, 248-9) and Polyphen2_HVAR (Adzhubei et al., Nat. Methods, 2010, 7, 248-9), LRT (Chun et al., Genome Res., 2009, 19, 1553-61), and MutationTaster (Schwarz et al., Nat. Methods, 2010, 7, 575-6). For each gene, inclusion in these seven gene burden exposures was determined by the alternative allele frequency (AAF) and functional annotation of each variant: 1) pLOF variants with AAF<1%; 2) pLOF or missense variants predicted as deleterious by 5 / 5 algorithms with AAF<1%; 3) pLOF or missense variants predicted as deleterious by 5 / 5 algorithms with AAF<0.1%; 4) pLOF or missense variants predicted as deleterious by at least 1 / 5 algorithms with AAF<1%; 5) pLOF or missense variants predicted as deleterious by at least 1 / 5 algorithms with AAF<0.1%; 6) pLOF or any missense with AAF<1%; 7) pLOF or any missense variant with AAF<0.1%.

[0251] Association analysis of rare pLOF and missense mutation gene burden Associations between the burden of rare predicted loss-of-function or missense variants in a given gene and phenotype were tested by fitting linear (for quantitative traits) or firth bias-corrected logistic (for binary traits) regression models adjusted for polygenic scores approximating the genomic kinship matrix using REGENIE v1.0 (see World Wide Web at doi.org / 10.1101 / 2020.06.19.162354 ). Analyses were stratified by ancestry and included age, 2 , gender, age and gender, and age 2 We adjusted for the interaction term between β and sex, experimental batch-related covariates, 10 common variant-derived principal components, and 20 rare variant-derived principal components. Results across cohorts for each variant-phenotype association were combined using fixed-effects inverse variance-weighted meta-analysis. In gene burden testing, all individuals are labeled as heterozygous if they have one or more eligible rare variants (as described above based on frequency and functional annotation) and as homozygous if they have any eligible variant in the homozygous state. This "composite genotype" is then used to test for association. Linear interaction models were fitted to the same analytical approach.

[0252] In addition to those described herein, various modifications of the described subject matter will become 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, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. A composition for treating a subject having a liver disease, the composition comprising a CAMP-responsive element-binding protein 3-like 3 (CREB3L3) inhibitor.

2. The composition according to claim 1, wherein the liver disease includes a parenchymal liver disease, liver fibrosis, cirrhosis, or non-alcoholic fatty liver disease (NAFLD).

3. The composition according to claim 1, wherein the CREB3L3 inhibitor comprises an inhibitory nucleic acid molecule.

4. The composition according to claim 3, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with a CREB3L3 nucleic acid molecule.

5. The composition according to any one of claims 1 to 4, wherein the subject is a CREB3L3 standard.

6. The composition according to any one of claims 1 to 4, wherein the subject is heterozygous for a CREB3L3 variant nucleic acid molecule.

7. The composition according to claim 6, wherein the CREB3L3 variant nucleic acid molecule encodes Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, Asp182Asn-D, or Asp181Asn.

8. The composition according to claim 6, wherein the CREB3L3 variant nucleic acid molecule encodes Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, or Asp182Asn-D.

9. The CREB3L3 variant nucleic acid molecule is: A genomic nucleic acid molecule having a nucleotide sequence containing adenine at a position corresponding to position 6,120 described in SEQ ID NO: 2; An mRNA molecule having a nucleotide sequence containing adenine at a position corresponding to position 661 described in SEQ ID NO: 17, position 649 described in SEQ ID NO: 18, position 624 described in SEQ ID NO: 19, position 624 described in SEQ ID NO: 20, position 658 described in SEQ ID NO: 21, position 661 described in SEQ ID NO: 22, position 661 described in SEQ ID NO: 23, position 663 described in SEQ ID NO: 24, position 660 described in SEQ ID NO: 25, position 649 described in SEQ ID NO: 26, position 624 described in SEQ ID NO: 27, or position 691 described in SEQ ID NO: 28; or A cDNA molecule having a nucleotide sequence containing adenine at a position corresponding to the 661st position described in SEQ ID NO: 45, the 649th position described in SEQ ID NO: 46, the 624th position described in SEQ ID NO: 47, the 624th position described in SEQ ID NO: 48, the 658th position described in SEQ ID NO: 49, the 661st position described in SEQ ID NO: 50, the 661st position described in SEQ ID NO: 51, the 663rd position described in SEQ ID NO: 52, the 660th position described in SEQ ID NO: 53, the 649th position described in SEQ ID NO: 54, the 624th position described in SEQ ID NO: 55, or the 691st position described in SEQ ID NO:

56. The composition according to claim 7. **Claim 10** A composition for treating a subject having a liver disease, comprising a therapeutic agent for treating the liver disease, wherein the treatment comprises: determining whether the subject has a CREB3L3 mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CAMP-responsive element-binding protein 3-like 3 (CREB3L3); obtaining or having obtained a biological sample from the subject; performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype comprising the CREB3L3 mutant nucleic acid molecule encoding the loss-of-function polypeptide predicted by CREB3L3; administering or continuing to administer the therapeutic agent for treating the liver disease at a standard dosage to a subject who is a CREB3L3 reference, and administering a CREB3L3 inhibitor to the subject; administering or continuing to administer the therapeutic agent for treating the liver disease in an amount equal to or less than the standard dosage to a subject who is heterozygous for the CREB3L3 mutant nucleic acid molecule, and administering a CREB3L3 inhibitor to the subject; wherein the presence of a genotype having the CREB3L3 mutant nucleic acid molecule encoding the loss-of-function polypeptide predicted by CREB3L3 indicates that the subject has a low risk of developing the liver disease. **Claim 11** The composition according to claim 10, wherein the subject is a CREB3L3 reference, and the subject is administered or continues to be administered the therapeutic agent for treating the liver disease at a standard dosage and is administered a CREB3L3 inhibitor. **Claim 12** The composition according to claim 10, wherein the subject is heterozygous for a CREB3L3 variant nucleic acid molecule, and the subject is administered, or continues to be administered, a therapeutic agent for treating the liver disease in an amount equal to or less than the standard dosage, and is administered a CREB3L3 inhibitor.

13. The composition according to claim 12, wherein the CREB3L3 variant nucleic acid molecule encodes Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, Asp182Asn-D, or Asp181Asn.

14. The composition according to claim 12, wherein the CREB3L3 variant nucleic acid molecule encodes Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, or Asp182Asn-D.

15. The CREB3L3 variant nucleic acid molecule is: a genomic nucleic acid molecule having a nucleotide sequence containing adenine at a position corresponding to position 6,120 described in SEQ ID NO: 2; an mRNA molecule having a nucleotide sequence containing adenine at a position corresponding to position 661 described in SEQ ID NO: 17, position 649 described in SEQ ID NO: 18, position 624 described in SEQ ID NO: 19, position 624 described in SEQ ID NO: 20, position 658 described in SEQ ID NO: 21, position 661 described in SEQ ID NO: 22, position 661 described in SEQ ID NO: 23, position 663 described in SEQ ID NO: 24, position 660 described in SEQ ID NO: 25, position 649 described in SEQ ID NO: 26, position 624 described in SEQ ID NO: 27, or position 691 described in SEQ ID NO: 28; or a cDNA molecule generated from an mRNA molecule having a nucleotide sequence containing adenine at a position corresponding to position 661 described in SEQ ID NO: 45, position 649 described in SEQ ID NO: 46, position 624 described in SEQ ID NO: 47, position 624 described in SEQ ID NO: 48, position 658 described in SEQ ID NO: 49, position 661 described in SEQ ID NO: 50, position 661 described in SEQ ID NO: 51, position 663 described in SEQ ID NO: 52, position 660 described in SEQ ID NO: 53, position 649 described in SEQ ID NO: 54, position 624 described in SEQ ID NO: 55, or position 691 described in SEQ ID NO: 56, The composition according to claim 13.

16. The composition according to any one of claims 10 to 15, wherein the CREB3L3 inhibitor comprises an inhibitory nucleic acid molecule.

17. The composition according to claim 16, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule that hybridizes with a CREB3L3 nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).

18. A method for identifying a subject having a high risk of developing a liver disease, the method comprising: determining, or having determined, the presence or absence of a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by cAMP-responsive element-binding protein 3-like 3 (CREB3L3) in a biological sample obtained from the subject; wherein when the subject is a CREB3L3 standard, the subject has a high risk of developing the liver disease; and when the subject is heterozygous or homozygous for a CREB3L3 variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3, the subject has a low risk of developing the liver disease.

19. The method according to claim 18, wherein the CREB3L3 variant nucleic acid molecule encodes Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, Asp182Asn-D, or Asp181Asn.

20. The method according to claim 18, wherein the CREB3L3 variant nucleic acid molecule encodes Asp182Asn-A, Asp182Asn-B, Asp182Asn-C, or Asp182Asn-D.

21. The CREB3L3 variant nucleic acid molecule is: a genomic nucleic acid molecule having a nucleotide sequence containing adenine at a position corresponding to position 6,120 described in SEQ ID NO: 2; an mRNA molecule having a nucleotide sequence containing adenine at a position corresponding to position 661 described in SEQ ID NO: 17, position 649 described in SEQ ID NO: 18, position 624 described in SEQ ID NO: 19, position 624 described in SEQ ID NO: 20, position 658 described in SEQ ID NO: 21, position 661 described in SEQ ID NO: 22, position 661 described in SEQ ID NO: 23, position 663 described in SEQ ID NO: 24, position 660 described in SEQ ID NO: 25, position 649 described in SEQ ID NO: 26, position 624 described in SEQ ID NO: 27, or position 691 described in SEQ ID NO: 28; or A cDNA molecule generated from an mRNA molecule, having a nucleotide sequence containing adenine at a position corresponding to position 661 described in SEQ ID NO: 45, position 649 described in SEQ ID NO: 46, position 624 described in SEQ ID NO: 47, position 624 described in SEQ ID NO: 48, position 658 described in SEQ ID NO: 49, position 661 described in SEQ ID NO: 50, position 661 described in SEQ ID NO: 51, position 663 described in SEQ ID NO: 52, position 660 described in SEQ ID NO: 53, position 649 described in SEQ ID NO: 54, position 624 described in SEQ ID NO: 55, or position 691 described in SEQ ID NO:

56. The method according to claim 19. [

22. ] Use of a therapeutic agent for treating liver disease in the preparation of a medicament for treating liver disease in a subject, wherein the subject A genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by cAMP-responsive element-binding protein 3-like 3 (CREB3L3), having a nucleotide sequence containing adenine or its complement at a position corresponding to position 6,120 described in SEQ ID NO: 2; Adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 17; adenine or its complement at a position corresponding to position 649 described in SEQ ID NO: 18; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 19; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 20; adenine or its complement at a position corresponding to position 658 described in SEQ ID NO: 21; adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 22; adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 23; adenine or its complement at a position corresponding to position 663 described in SEQ ID NO: 24; adenine or its complement at a position corresponding to position 660 described in SEQ ID NO: 25; adenine or its complement at a position corresponding to position 649 described in SEQ ID NO: 26; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 27; or adenine or its complement at a position corresponding to position 691 described in SEQ ID NO: 28, an mRNA molecule encoding a loss-of-function polypeptide predicted by CREB3L3 or its complement; or A cDNA molecule encoding a loss-of-function polypeptide predicted by CREB3L3, or its complement, having a nucleotide sequence comprising adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 45; adenine or its complement at a position corresponding to position 649 described in SEQ ID NO: 46; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 47; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 48; adenine or its complement at a position corresponding to position 658 described in SEQ ID NO: 49; adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 50; adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 51; adenine or its complement at a position corresponding to position 663 described in SEQ ID NO: 52; adenine or its complement at a position corresponding to position 660 described in SEQ ID NO: 53; adenine or its complement at a position corresponding to position 649 described in SEQ ID NO: 54; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 55; or adenine or its complement at a position corresponding to position 691 described in SEQ ID NO:

56. **Claim 23** Use of a CREB3L3 inhibitor in the preparation of a medicament for the treatment of liver disease in a subject, wherein the subject is a) a reference for a CREB3L3 genomic nucleic acid molecule, a CREB3L3 mRNA molecule, or a CREB3L3 cDNA molecule, or b) i) a genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by CREB3L3, or its complement, having a nucleotide sequence comprising adenine or its complement at a position corresponding to position 6,120 described in SEQ ID NO: 2; ii) an mRNA molecule or its complement encoding a loss-of-function polypeptide predicted by CREB3L3, having a nucleotide sequence comprising adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 17; adenine or its complement at a position corresponding to position 649 described in SEQ ID NO: 18; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 19; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 20; adenine or its complement at a position corresponding to position 658 described in SEQ ID NO: 21; adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 22; adenine or its complement at a position corresponding to position 661 described in SEQ ID NO: 23; adenine or its complement at a position corresponding to position 663 described in SEQ ID NO: 24; adenine or its complement at a position corresponding to position 660 described in SEQ ID NO: 25; adenine or its complement at a position corresponding to position 649 described in SEQ ID NO: 26; adenine or its complement at a position corresponding to position 624 described in SEQ ID NO: 27; or adenine or its complement at a position corresponding to position 691 described in SEQ ID NO: 28; or iii) Adenine, or its complement, at the position corresponding to position 661 described in SEQ ID NO: 45; adenine, or its complement, at the position corresponding to position 649 described in SEQ ID NO: 46; adenine, or its complement, at the position corresponding to position 624 described in SEQ ID NO: 47; adenine, or its complement, at the position corresponding to position 624 described in SEQ ID NO: 48; adenine, or its complement, at the position corresponding to position 658 described in SEQ ID NO: 49; adenine, or its complement, at the position corresponding to position 661 described in SEQ ID NO: 50; adenine, or its complement, at the position corresponding to position 661 described in SEQ ID NO: 51; adenine, or its complement, at the position corresponding to position 663 described in SEQ ID NO: 52; adenine, or its complement, at the position corresponding to position 660 described in SEQ ID NO: 53; adenine, or its complement, at the position corresponding to position 649 described in SEQ ID NO: 54; adenine, or its complement, at the position corresponding to position 624 described in SEQ ID NO: 55; or adenine, or its complement, at the position corresponding to position 691 described in SEQ ID NO: 56, for use in a heterozygous form, of a cDNA molecule encoding a loss-of-function polypeptide predicted by CREB3L3 or its complement.

24. The use according to claim 23, wherein the CREB3L3 inhibitor comprises an inhibitory nucleic acid molecule.

25. The use according to claim 24, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes with a CREB3L3 nucleic acid molecule.