Hydroxysteroid 17-β dehydrogenase 13 (HSD17b13) variants and uses thereof

Abstract

To provide a method for determining a subject's susceptibility to developing a liver disease or for diagnosing a subject with liver disease.SOLUTION: Provided is a method for detecting a variant HSD17B13 gene in a human subject. The method comprises or consists of performing an assay on a biological sample collected from the human subject, wherein the assay determines whether a thymine is inserted between positions corresponding to positions 12665 and 12666 of a specific sequence of a wild-type HSD17B13 gene or whether a thymine is present at a position corresponding to position 12666 of another specific sequence of a variant HSD17B13 gene, and wherein the presence of the thymine indicates the variant HSD17B13 gene.SELECTED DRAWING: Figure 1A-1

Description

[Technical Field]

[0001] This application contains a Sequence Listing that has been submitted electronically as a text file of 147 kilobytes in size, entitled 18923800802SEQ, created on January 18, 2018. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates generally to the field of genetics. More particularly, the present disclosure relates to genetic alterations and polypeptide variants in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13), for example, that are associated with liver disease. [Background technology]

[0003] Throughout this specification, various references are cited, including patents, patent applications, accession numbers, technical papers and journal articles. Each reference is incorporated herein by reference in its entirety for all purposes.

[0004] 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 all deaths) (Non-Patent Document 1). The most common causes of cirrhosis in the United States are alcoholic liver disease, chronic hepatitis C, and non-alcoholic fatty liver disease (NAFLD), which accounted for approximately 80% of patients awaiting liver transplantation between 2004 and 2013 (Non-Patent Document 2). The estimated prevalence of NAFLD in the United States ranges from 19 to 46 percent (Non-Patent Documents 3-5), and has been increasing over time, likely in conjunction with rising obesity rates, a major risk factor for NAFLD (Non-Patent Document 6) (Non-Patent Document 7). While significant advances have been made in the treatment of hepatitis C (Non-Patent Documents 8-9), there are currently no evidence-based treatments for alcoholic or non-alcoholic liver disease or cirrhosis.

[0005] Previous genome-wide association studies (GWAS) have identified a limited number of genes and variants associated with chronic liver disease. The most clearly documented genetic association to date is for a common missense variant in the patatin-like phospholipase domain-containing 3 gene (PNPLA3 p.Ile148Met, rs738409). This variant was first found to be associated with increased risk of nonalcoholic fatty liver disease (NAFLD) (Non-Patent Documents 10-11), and subsequently with disease severity (Non-Patent Documents 12-13) and progression (Non-Patent Document 14). Alterations in the transmembrane 6 superfamily member 2 (TM6SF2) gene have also been shown to increase NAFLD risk (Non-Patent Documents 15-17). While the normal functions of these two proteins are not fully understood, both have been proposed to be involved in hepatocyte lipid metabolism. The mechanisms by which PNPLA3 and TM6SF2 variants contribute to increased liver disease risk remain unclear. GWAS have also identified several genetic factors associated with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), quantitative markers of hepatocellular injury and hepatic fat accumulation that are frequently measured clinically (Non-Patent Documents 18-19). To date, no protective genetic variants have been shown for chronic liver disease. Protective genetic variants in other conditions (e.g., loss-of-function variants of PCSK9 that reduce cardiovascular disease risk) have led to the development of new classes of therapeutic agents.

[0006] Identifying the genetic factors underlying the onset and progression of chronic liver disease could improve risk stratification and lay the foundation for novel therapeutic strategies. A better understanding of the underlying genetic factors is needed to improve risk stratification and develop novel therapies for liver disease. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kochanek et al.,Natl.Vital Stat.Rep.,2016,65,1-122 [Non-licensed document 2] Wong et al.,Gastroenterology,2015,148,547-555 [Non-licensed document 3] Browning et al.,Hepatology,2004,40,1387-1395

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Non-licensed literature 9

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[0008] The present disclosure provides novel HSD17B13 variants that will aid in the understanding of HSD17B13 biology and facilitate the diagnosis and treatment of subjects with liver disease. The present disclosure provides nucleic acid molecules, polypeptides, probes, primers, compositions and methods related to variant HSD17B13 rs72613567 genes, variant HSD17B13 transcripts and variant HSD17B13 protein isoforms.

[0009] The present disclosure also provides nucleic acid molecules encoding variant HSD17B13 protein isoforms. In some embodiments, the nucleic acid molecules encode variant HSD17B13 protein isoform C, isoform D, isoform F, isoform G, or isoform H. In some embodiments, the nucleic acid molecules encode variant HSD17B13 protein isoform D.

[0010] The present disclosure also provides a nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides of the HSD17B13 gene, which contiguous nucleotides are at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequence in SEQ ID NO:2, and having a thymine at a position corresponding to position 12666 of SEQ ID NO:2.

[0011] The present disclosure also provides nucleic acid molecules comprising or consisting of a nucleotide sequence encoding a polypeptide having an amino acid sequence that is 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 the amino acid sequence of HSD17B13 isoform D (SEQ ID NO:42). In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of HSD17B13 isoform D (SEQ ID NO:42). In some embodiments, the nucleic acid molecule of the present disclosure comprises or consists of a nucleotide sequence that is 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 the nucleotide sequence of HSD17B13 transcript D (SEQ ID NO:6, 15, 24, or 33). In some embodiments, the nucleic acid molecule of the present disclosure is RNA and its cDNA comprises or consists of SEQ ID NO:6, or comprises or consists of SEQ ID NO:24, or the nucleic acid molecule is mRNA and its cDNA comprises or consists of SEQ ID NO:15, or comprises or consists of SEQ ID NO:33.

[0012] The disclosure also provides nucleic acid molecules (including probes and primers such as variation-specific probes or variation-specific primers) that hybridize to or near the variation in a variant HSD17B13 rs72613567 gene or HSD17B13 transcript.

[0013] The present disclosure also provides a nucleic acid molecule comprising or consisting of about 5 nucleotides up to about 50 nucleotides that specifically hybridizes to a variant HSD17B13 gene or its complement in a region that includes a position corresponding to position 12666 of SEQ ID NO:2, wherein the nucleic acid molecule specifically hybridizes to a variant HSD17B13 gene having a thymine at a position corresponding to position 12666 of SEQ ID NO:2, or its complement.

[0014] The present disclosure also provides a nucleic acid molecule comprising or consisting of about 5 nucleotides up to about 50 nucleotides that specifically hybridizes to variant HSD17B13 transcript D, wherein the nucleic acid molecule specifically hybridizes to i) a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 6, 15, 24 or 33, or ii) the complement of the nucleotide sequence of i).

[0015] The present disclosure also provides nucleic acid molecules comprising, or alternatively consisting of, about 5 to up to about 50 nucleotides, including i) a nucleic acid molecule that specifically hybridizes to a nucleotide sequence 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 the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or its complement, ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D, and / or iii) a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of transcript D. In some embodiments, the nucleic acid molecule of the present disclosure specifically hybridizes to an RNA molecule comprising or consisting of SEQ ID NO: 6, or its cDNA comprising or consisting of SEQ ID NO: 24, or the nucleic acid molecule specifically hybridizes to an mRNA comprising or consisting of SEQ ID NO: 15, a cDNA comprising or consisting of SEQ ID NO: 33, or their complements. In some embodiments, the nucleic acid molecules of the present disclosure are linked to a heterologous nucleic acid or comprise a heterologous label.

[0016] The disclosure also provides vectors comprising any of these nucleic acid molecules. The disclosure also provides cells containing any of these nucleic acid molecules. The present disclosure also provides cells containing any of these vectors.

[0017] The disclosure also provides compositions comprising any of these nucleic acid molecules. The present disclosure also provides compositions comprising any of these vectors. The present disclosure also provides compositions comprising any of these cells.

[0018] The present disclosure also provides for the use of any of these nucleic acid molecules to detect variant HSD17B13 genes or transcripts, to determine a human subject's susceptibility or risk for developing liver disease, or to determine a human subject's risk for progressing to a clinically more advanced stage of fatty liver disease.

[0019] The present disclosure also provides polypeptides corresponding to each of the different variant HSD17B13 protein isoforms. The present disclosure also provides polypeptides comprising or consisting of an amino acid sequence that is 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 the amino acid sequence of HSD17B13 isoform D (SEQ ID NO: 42). In some embodiments, a polypeptide of the present disclosure comprises or consists of the amino acid sequence of SEQ ID NO: 42. In some embodiments, a polypeptide of the present disclosure is linked to a heterologous molecule.

[0020] The disclosure also provides compositions comprising any of these polypeptides. The present disclosure also provides methods for detecting variant HSD17B13 rs72613567 genes, variant HSD17B13 transcripts (such as transcript D), and variant HSD17B13 isoforms (such as isoform D).

[0021] The disclosure also provides a method for detecting a variant HSD17B13 gene in a human subject, comprising performing an assay on a biological sample from the human subject, the assay determining whether a wild-type HSD17B13 gene has a thymine inserted between positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1, or whether a variant HSD17B13 gene has a thymine at a position corresponding to position 12666 of SEQ ID NO:2, wherein the presence of a thymine indicates a variant HSD17B13 gene. In some embodiments, the assay comprises sequencing a portion of the HSD17B13 gene that includes positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1, or that includes a position corresponding to position 12666 of SEQ ID NO:2. In some embodiments, the assay comprises: i) contacting the biological sample with primers that hybridize to a region of the HSD17B13 gene that is within 50 nucleotides of a position corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1 in the wild-type HSD17B13 gene, or within 50 nucleotides of a position corresponding to position 12666 of SEQ ID NO: 2 in the variant HSD17B13 gene; and ii) contacting the biological sample with primers that hybridize to a region of the HSD17B13 gene that is within 50 nucleotides of a position corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1 in the wild-type HSD17B13 gene. or a position corresponding to position 12666 of SEQ ID NO: 2 in the variant HSD17B13 gene; and iii) determining whether the extension product of the primer contains a thymine inserted between positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1 in the wild-type HSD17B13 gene or a thymine is present at a position corresponding to position 12666 of SEQ ID NO: 2 in the variant HSD17B13 gene. In some embodiments, the method further comprises determining whether the human subject is homozygous for the variant HSD17B13 gene.

[0022] The present disclosure also provides methods for detecting the presence of HSD17B13 transcript D in a human subject, comprising performing an assay on a biological sample obtained from the subject, whereby the assay determines the presence of HSD17B13 transcript D in the biological sample. In some embodiments, the assay comprises or consists of contacting the biological sample with one or more primers or probes that specifically hybridize to a nucleic acid sequence of HSD17B13 transcript D or its complement, and determining whether hybridization has occurred. In some embodiments, the method further comprises, or consists of, specifically detecting transcript D by using a nucleic acid molecule comprising or consisting of about 5 to up to about 50 nucleotides comprising or consisting of: i) a nucleic acid molecule that specifically hybridizes to a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or its complement; ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D; and / or iii) a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of transcript D. In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the one or more primers or probes specifically hybridize to SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, and / or SEQ ID NO: 33. In some embodiments, the assay further comprises using primers or probes that specifically hybridize to one or more of transcripts A, B, C, E, F, F', G, and / or H but do not specifically hybridize to transcript D, and determining that no hybridization occurred. In some embodiments, the assay comprises reverse transcription polymerase chain reaction (RT-PCR).In some embodiments, the assay comprises sequencing.

[0023] The present disclosure also provides a method for detecting the presence of HSD17B13 isoform D in a human subject, comprising performing an assay on a biological sample taken from the human subject, wherein the assay determines the presence of HSD17B13 isoform D in the biological sample. In some embodiments, HSD17B13 isoform D comprises or consists of an amino acid sequence that is 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 SEQ ID NO: 42. In some embodiments, the assay comprises sequencing.

[0024] The present disclosure also provides methods for determining a subject's susceptibility to developing liver disease and / or diagnosing a subject for liver disease by determining the presence of a variant HSD17B13 rs72613567 gene, a variant HSD17B13 transcript (such as transcript D), and a variant HSD17B13 isoform (such as isoform D) in a sample obtained from the subject.

[0025] The disclosure provides a method for determining a human subject's susceptibility or risk for developing liver disease, comprising: a) performing an assay on a biological sample from the human subject, the assay determining whether a thymine is present in the HSD17B13 gene at positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1, or whether a thymine is present in a variant HSD17B13 gene at position 12666 of SEQ ID NO: 2; and b) determining whether a thymine is present in the wild-type HSD17B13 gene at positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1, or whether a thymine is present in the variant HSD17B13 gene at position 12666 of SEQ ID NO: 2. or classifying the human subject as having a reduced risk of developing liver disease if a thymine is present in a variant HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2, or as having an elevated risk of developing liver disease if a thymine is not inserted between positions 12665 ​​and 12666 of SEQ ID NO: 1 in the HSD17B13 gene or if a thymine is not present in a variant HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2. In some embodiments, the liver disease is a chronic liver disease. In some embodiments, the liver disease is selected from the group consisting of fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple steatosis, steatohepatitis, fibrosis, and non-alcoholic steatohepatitis (NASH).In some embodiments, the assay comprises, or consists of: i) contacting the biological sample with a primer that hybridizes to a region of the HSD17B13 gene that is within 50 nucleotides of a position in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or 12666 of SEQ ID NO:2; ii) extending the primer to at least pass through a position in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or 12666 of SEQ ID NO:2; and iii) determining whether the extension product of the primer contains a thymine inserted between positions 12665 ​​and 12666 of SEQ ID NO:1 in the wild-type HSD17B13 gene or a thymine present at a position corresponding to position 12666 of SEQ ID NO:2 in the variant HSD17B13 gene. In some embodiments, the assay comprises or consists of contacting the biological sample with a primer or probe that specifically hybridizes under stringent conditions to a variant HSD17B13 gene having a thymine at position 12666 of SEQ ID NO:2, but does not hybridize to the corresponding wild-type HSD17B13 gene, and determining whether hybridization has occurred. In some embodiments, the variant HSD17B13 gene is detected by sequencing. In some embodiments, the method further comprises determining whether the human subject is homozygous for the variant HSD17B13 gene.

[0026] The present disclosure also provides methods for determining a human subject's susceptibility or risk for developing liver disease, comprising or consisting of: a) performing an assay on a biological sample obtained from the human subject, wherein the assay determines the presence of HSD17B13 transcript D in the biological sample; and b) classifying the human subject as having a reduced risk for developing liver disease if HSD17B13 transcript D is present in the biological sample, or as having an elevated risk for developing liver disease if HSD17B13 transcript D is absent from the biological sample. In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the HSD17B13 transcript D is RNA and comprises or consists of SEQ ID NO:6, or a cDNA thereof comprising or consisting of SEQ ID NO:24, or the HSD17B13 transcript D is mRNA and comprises or consists of SEQ ID NO:15, or a cDNA thereof comprising or consisting of SEQ ID NO:33. In some embodiments, the assay determines the expression level of HSD17B13 transcript D in the biological sample, wherein an elevated expression level of HSD17B13 transcript D compared to a control sample obtained from a control human subject homozygous for the wild-type HSD17B13 allele indicates a decreased risk of developing liver disease, and a similar or decreased expression level of HSD17B13 transcript D compared to the control sample indicates an increased risk of developing liver disease. In some embodiments, the liver disease is chronic liver disease.In some embodiments, the liver disease is selected from the group consisting of fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple steatosis, steatohepatitis, fibrosis, and non-alcoholic steatohepatitis (NASH). In some embodiments, the assay comprises or consists of contacting the biological sample with one or more primers or probes that specifically hybridize to a nucleic acid sequence of HSD17B13 transcript D or its complement, and determining whether hybridization has occurred. In some embodiments, the method further comprises specifically detecting transcript D by using a nucleic acid molecule comprising from about 5 nucleotides up to about 50 nucleotides comprising: i) a nucleic acid molecule that specifically hybridizes to a nucleotide sequence 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 the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or its complement; ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D; and / or iii) a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of transcript D. In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the one or more primers or probes specifically hybridize to SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24, and / or SEQ ID NO:33. In some embodiments, the assay further comprises using primers or probes that specifically hybridize to one or more of transcripts A, B, C, E, F, F', G, and / or H, but not to transcript D, and determining that no hybridization occurred. In some embodiments, the assay comprises reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR).In some embodiments, the assay comprises sequencing.

[0027] The present disclosure also provides a method for determining a human subject's susceptibility or risk for developing liver disease, comprising or consisting of: a) detecting the presence of HSD17B13 isoform D in a biological sample obtained from the human subject; and b) classifying the human subject as having a reduced risk for developing liver disease if HSD17B13 isoform D is detected in the biological sample, or classifying the human subject as having a reduced risk for developing liver disease if HSD17B13 isoform D is not detected in the biological sample. In some embodiments, HSD17B13 isoform D comprises or consists of an amino acid sequence that is 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 SEQ ID NO: 42. In some embodiments, the liver disease is chronic liver disease. In some embodiments, the liver disease is selected from the group consisting of fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple steatosis, steatohepatitis, fibrosis, and non-alcoholic steatohepatitis (NASH). In some embodiments, the detecting comprises sequencing.

[0028] The disclosure provides a method for determining the risk of a human subject progressing to a clinically more advanced stage of fatty liver disease, comprising: a) performing an assay on a biological sample from the human subject, wherein the assay determines whether a thymine is present between positions 12665 ​​and 12666 of SEQ ID NO: 1 in a wild-type HSD17B13 gene or a thymine is present at position 12666 of SEQ ID NO: 2 in a variant HSD17B13 gene; and b) determining whether a thymine is present between positions 12665 ​​and 12666 of SEQ ID NO: 1 in a wild-type HSD17B13 gene or a thymine is present at position 12666 of SEQ ID NO: 2 in a variant HSD17B13 gene. The present invention also provides a method for determining whether a human subject has a reduced risk of progressing to a clinically more advanced stage of fatty liver disease if a thymine is present in the gene at position corresponding to position 12666 of SEQ ID NO:2, or if a human subject has an increased risk of progressing to a clinically more advanced stage of fatty liver disease if there is no thymine inserted in the HSD17B13 gene between positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1, or if there is no thymine present in the variant HSD17B13 gene at position corresponding to position 12666 of SEQ ID NO:2. In some embodiments, the assay comprises, or consists of: i) contacting the biological sample with a primer that hybridizes to a region of the HSD17B13 gene that is within 50 nucleotides of a position in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or 12666 of SEQ ID NO:2; ii) extending the primer to at least pass through a position in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or 12666 of SEQ ID NO:2; and iii) determining whether the extension product of the primer contains a thymine inserted between positions 12665 ​​and 12666 of SEQ ID NO:1 in the wild-type HSD17B13 gene or a thymine present at a position corresponding to position 12666 of SEQ ID NO:2 in the variant HSD17B13 gene.In some embodiments, the assay comprises or consists of contacting the biological sample with a primer or probe that specifically hybridizes under stringent conditions to a variant HSD17B13 gene having a thymine at position 12666 of SEQ ID NO:2, but does not specifically hybridize to the corresponding wild-type HSD17B13 gene, and determining whether hybridization has occurred. In some embodiments, the variant HSD17B13 gene is detected by sequencing. In some embodiments, the method further comprises determining whether the human subject is homozygous for the variant HSD17B13 gene.

[0029] The present disclosure also provides a method for determining a human subject's risk of developing a clinically more advanced stage of fatty liver disease, comprising: a) performing an assay on a biological sample taken from the human subject, wherein the assay determines the presence of HSD17B13 transcript D in the biological sample; and b) classifying the human subject as having a reduced risk of developing a clinically more advanced stage of fatty liver disease if HSD17B13 transcript D is present in the biological sample, or as having an increased risk of developing a clinically more advanced stage of fatty liver disease if HSD17B13 transcript D is absent from the biological sample. In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the HSD17B13 transcript D is RNA and comprises or consists of SEQ ID NO:6, or a cDNA thereof that comprises or consists of SEQ ID NO:24, or the HSD17B13 transcript D is mRNA and comprises or consists of SEQ ID NO:15, or a cDNA thereof that comprises or consists of SEQ ID NO:33. In some embodiments, the assay determines the expression level of HSD17B13 transcript D in the biological sample, wherein an elevated expression level of HSD17B13 transcript D compared to a control sample obtained from a control human subject homozygous for the wild-type HSD17B13 allele indicates a decreased risk of progressing to a more clinically advanced stage of fatty liver disease, and a similar or decreased expression level of HSD17B13 transcript D compared to the control sample indicates an increased risk of progressing to a more clinically advanced stage of fatty liver disease.In some embodiments, the assay comprises or consists of contacting the biological sample with one or more primers or probes that specifically hybridize to a nucleic acid sequence of HSD17B13 transcript D or its complement, and determining whether hybridization has occurred. In some embodiments, the method further comprises specifically detecting transcript D by using a nucleic acid molecule comprising from about 5 nucleotides up to about 50 nucleotides, comprising: i) a nucleic acid molecule that specifically hybridizes to a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or its complement; ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D; and / or iii) a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of transcript D. In some embodiments, the one or more primers or probes specifically hybridize to SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24, and / or SEQ ID NO:33. In some embodiments, the assay further comprises using primers or probes that specifically hybridize to one or more of transcripts A, B, C, E, F, F', G, and / or H, but not to transcript D, and determining that no hybridization occurred. In some embodiments, the assay comprises reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). In some embodiments, the assay comprises sequencing.

[0030] The present disclosure also provides a method for determining a human subject's risk of progressing to a clinically more advanced stage of fatty liver disease, comprising: a) detecting the presence of HSD17B13 isoform D in a biological sample obtained from the human subject; and b) classifying the human subject as having a reduced risk of progressing to a clinically more advanced stage of liver disease if HSD17B13 isoform D is detected in the biological sample. In some embodiments, HSD17B13 isoform D comprises an amino acid sequence that is 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 SEQ ID NO: 42. In some embodiments, the detection step comprises sequencing.

[0031] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the disclosure. [Brief explanation of the drawings]

[0032] [Figure 1A-1] Manhattan plot (left) and quantile-quantile plot (right) of the association between single nucleotide variants and serum transaminase levels in the GHS discovery cohort are shown, showing genetic variants significantly associated with alanine aminotransferase (ALT) levels at P < 1.0 × 10-7. [Figure 1A-2] Same as above. [Figure 1B-1] Manhattan plot (left) and quantile-quantile plot (right) of the association between single nucleotide variants and serum transaminase levels in the GHS discovery cohort are shown, showing genetic variants significantly associated with aspartate aminotransferase (AST) levels at P < 1.0 × 10-7. The association analysis was well calibrated, as indicated by the exome-wide quantile-quantile plot and lambda values ​​for genomic controls. [Figure 1B-2]Same as above. [Figure 2A] HSD17B13 rs72613567:TA is associated with a reduced risk of alcoholic and non-alcoholic liver disease phenotypes. HSD17B13 rs72613567 was associated with reduced odds ratios for non-alcoholic liver disease, alcoholic liver disease, cirrhosis, and hepatocellular carcinoma in an allele dose-dependent manner. These odds ratios were calculated using logistic regression, adjusting for age, age squared, sex, BMI, and principal components of ancestry. Heterozygous carrier genotype odds ratios (Het OR) and homozygous carrier genotype odds ratios (Hom OR) are also shown. [Figure 2B] We demonstrate that HSD17B13 rs72613567:TA is associated with a reduced risk of alcoholic and nonalcoholic liver disease phenotypes. In the Dallas Liver Study, HSD17B13 rs72613567 was associated with reduced odds ratios for all liver disease in an allele-dose-dependent manner. Similar allele-dose-dependent effects were observed across liver disease subtypes. Odds ratios were calculated using logistic regression, adjusting for age, age squared, sex, BMI, and self-reported ethnicity. [Figure 3A] HSD17B13 rs72613567:TA is associated with a reduced risk of progression from simple steatosis to steatohepatitis and fibrosis. Prevalence of histopathologically characterized liver disease by HSD17B13 rs72613567 genotype in 2,391 individuals with liver biopsy specimens from the GHS bariatric surgery cohort. The prevalence of normal liver did not appear to differ by genotype (P = 0.5 by chi-square test for trend in proportions), but each TA allele was associated with a reduced prevalence of NASH (P = 1.6 × 10-4) and an increased prevalence of simple steatosis (P = 1.1 × 10-3). [Figure 3B]HSD17B13 rs72613567:TA was associated with a reduced risk of progression from simple steatosis to steatohepatitis and fibrosis. In the GHS bariatric surgery cohort, HSD17B13 rs72613567 was associated with a 13% reduced odds ratio for NASH and a 13% reduced odds ratio for fibrosis in heterozygous TA carriers and a 52% reduced odds ratio for NASH and a 61% reduced odds ratio for fibrosis in homozygous TA carriers. Odds ratios were calculated using logistic regression, adjusted for age, age squared, sex, BMI, and principal components of ancestry. Genotype odds ratios for heterozygous carriers (Het OR) and homozygous carriers (Hom OR) are also shown. [Figure 4A]The expression, subcellular localization, and enzymatic activity of novel HSD17B13 transcripts are shown. Figure 4A shows the expression levels of HSD17B13 transcripts A and D in carriers of the reference homozygous (T / T) splice variant, heterozygous (T / TA) variant, and homozygous (TA / TA) variant of the HSD17B13 rs72613567 splice variant. The coding region of the HSD17B13 gene is indicated by a vertical rectangle, untranslated regions are indicated by thick lines, and introns are indicated by thin lines. The asterisk in transcript D indicates the insertion of A from rs72613567. mRNA expression levels are shown in units of FPKM (fragments per kilobase of transcript per million mapped reads). Figure 4B shows Western blots of HSD17B13 from fresh-frozen human liver and HEK293 cell samples. Human liver samples were obtained from reference homozygous (T / T) carriers, variant heterozygous (T / TA) carriers, and variant homozygous (TA / TA) carriers of the HSD17B13 rs72613567 splice variant. Cell samples were obtained from HEK293 cells overexpressing untagged HSD17B13 transcripts A and D. HSD17B13 transcript D was translated into a truncated protein, IsoD, with a lower molecular weight than HSD17B13 IsoA. In Figure 4C, HSD17B13 IsoD protein levels were lower than IsoA protein levels in both human liver samples (left) and cell samples (right). Protein levels normalized to actin are shown in the bar graph. **P<0.001, *P<0.05. In Figure 4D, both HSD17B13 isoforms A and D were localized to lipid droplet membranes. HepG2 cells stably overexpressing HSD17B13 transcripts A or D were labeled with BODIPY to reveal lipid droplets and with anti-Myc to reveal HSD17B13 localization. All images are magnified to the same extent, and the scale bar indicates 10 μm. The inset shows a 4x magnification of the original image.Figure 4E shows the enzymatic activity of HSD17B13 isoforms A and D toward 17-β estradiol (estradiol), leukotriene B4 (LTB4), and 13-hydroxyoctadecadienoic acid (13(S)-HODE). HSD17B13 isoform D exhibited less than 10% of the enzymatic activity of isoform A. Figure 4F shows that overexpression of HSD17B13 isoform D in HEK293 cells resulted in limited conversion of estradiol (substrate) to estrone (product), as measured in the culture medium, whereas overexpressed HSD17B13 isoform A exhibited vigorous conversion. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 5A] Figure 1 shows a region association plot of alanine aminotransferase (ALT) levels in the region surrounding HSD17B13 in the GHS discovery cohort. [Figure 5B] This figure shows a regional association plot of aspartate aminotransferase (AST) levels in the region surrounding HSD17B13 in the GHS discovery cohort. Diamonds represent the splice variant rs72613567, and each circle represents a single-nucleotide variant. The color of the circle indicates linkage disequilibrium between the variant and rs72613567 (r2 calculated in the DiscovEHR cohort). Lines indicate estimated recombination rates in HapMap. The bottom panel shows the relative position and transcript strand of each gene at the locus. In the neighboring gene, HSD17B11, no significant association was found between ALT or AST and coding or splice region variants (most significant P values: 1.4 × 10-1 for ALT and 4.3 × 10-2 for AST). [Figure 6A]Figure 6A shows the mRNA expression levels of HSD17B13 transcript A in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Figure 6B shows the mRNA expression levels of HSD17B13 transcript B in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Figure 6C shows the mRNA expression levels of HSD17B13 transcript C in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Figure 6D shows the mRNA expression levels of HSD17B13 transcript D in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Figure 6E shows the mRNA expression levels of HSD17B13 transcript E in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Figure 6F shows the mRNA expression levels of HSD17B13 transcript F in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Figure 6G shows the mRNA expression levels of HSD17B13 transcript G in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Figure 6H shows the mRNA expression levels of HSD17B13 transcript H in carriers of the reference homozygous allele (T / T), carriers of the variant heterozygous allele (T / TA), and carriers of the variant homozygous allele (TA / TA). Each transcript is illustrated with its corresponding gene model.Coding regions of the gene model are shown as vertical rectangles, untranslated regions are shown as thick lines, introns are shown as thin lines, and an asterisk indicates the A insertion from rs72613567. Transcript expression levels differ by HSD17B13 genotype as shown by the box plots. mRNA expression levels are shown in FPKM (fragments per kilobase of transcript per million mapped reads). [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above. [Figure 6H] Same as above. [Figure 7A] Protein sequence alignment of all identified HSD17B13 isoforms (A-H) is shown. [Figure 7B] Protein sequence alignment of all identified HSD17B13 isoforms (A-H) is shown. [Figure 8A] HSD17B13 isoform D protein has been shown to have a low molecular weight and to be unstable when overexpressed in HEK293 cells. (Figure 8A) RT-PCR of HSD17B13 from HEK293 cells overexpressing HSD17B13 transcripts A (IsoA) and D (IsoD) showed that HSD17B13 IsoD RNA levels were higher than IsoA RNA levels. (Figure 8B) Western blot analysis from the same cell line showed that HSD17B13 transcript D was translated into a lower molecular weight truncated protein compared to HSD17B13 transcript A. (Figure 8C) IsoD RNA levels were higher than IsoA, but HSD17B13 IsoD protein levels were lower than IsoA protein levels. HSD17B13 protein levels were normalized to actin. *P<0.05. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9] These results demonstrate similar localization patterns of HSD17B13 isoform A and isoform D to isolated lipid droplets (LDs) from the HepG2 stable cell line. ADRP and TIP47 were used as lipid droplet markers. LAMP1 was used as a marker for the lysosomal compartment, calreticulin as a marker for the endoplasmic reticulum compartment, and COX IV as a marker for the mitochondrial compartment. GAPDH was included as a cytoplasmic marker, and actin was used as a cytoskeleton marker. This experiment was repeated twice in HepG2 cells, and the figure is representative of two runs. PNS = post-depletion fraction, TM = total membrane. [Figure 10A]We demonstrate that oleic acid increased triglyceride levels in HepG2 cells overexpressing HSD17B13 transcripts A or D. Figure 10A shows lipid droplets in both HepG2 cell lines expressing HSD17B13 transcript A and HSD17B13 transcript D treated with oleic acid to similar extents. Cells were labeled with BODIPY to show lipid droplets and anti-Myc to show HSD17B13 localization. Scale bars indicate 10 μm, and insets in each image represent 4x magnifications of the original images. Figure 10B shows that treatment with increasing concentrations of oleic acid increased triglyceride (TG) levels to similar extents in control (GFP-overexpressing cells), HSD17B13 transcript A cell lines, and HSD17B13 transcript D cell lines. In Figure 10C, the RNA levels of HSD17B13 transcripts A and D were comparable in the above cell lines. RNA levels are shown as reads per kilobase of transcript per million mapped reads (RPKM). Figure 10D shows a Western blot from HepG2 cells overexpressing HSD17B13 transcripts A and D. HSD17B13 transcript D was translated into a lower molecular weight truncated protein compared to HSD17B13 transcript A. In Figure 10E, HSD17B13 IsoD protein levels were lower than IsoA protein levels. Protein levels were normalized to actin. **P<0.01. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 11]The Km and Vmax values ​​for estradiol using purified recombinant HSD17B13 protein are shown. To determine Km and Vmax, assays were performed with 17β-estradiol at doses ranging from 0.2 μM to 200 μM, 500 μM NAD+, and 228 nM HSD17B13 at time points 5 to 180 min. Vmax and Km were then calculated using the Michaelis-Menten model and Prism software (GraphPad Software, USA). DETAILED DESCRIPTION OF THE INVENTION

[0033] Additional advantages of the present disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the embodiments disclosed herein. The advantages of the present disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments as claimed.

[0034] Throughout the specification and claims, various terms relating to aspects of the present disclosure are used. Unless otherwise indicated, such terms shall be given their ordinary meaning in the art. Other terms that are specifically defined shall be construed in a manner consistent with the definitions set forth herein.

[0035] Unless expressly stated otherwise, no method or embodiment set forth herein is intended to be construed as requiring its steps to be performed in a particular order. Thus, unless specifically stated by a method claim in the claims or description that steps should be limited to a particular order, no order is intended to be imposed in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or workflow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0036] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the terms "subject" and "patient" are used interchangeably. A subject may include any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs, sheep, etc.), pets (e.g., dogs, cats, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), and non-human primates (e.g., monkeys, ape-humans, etc.). In some embodiments, the subject is a human.

[0037] As used herein, "nucleic acid," "nucleic acid molecule," "nucleotide sequence," "polynucleotide," or "oligonucleotide" can include a polymeric form of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. One strand of a nucleic acid is also referred to as its complement.

[0038] Throughout this disclosure, the term "comprising" may be replaced with "consisting of" or "consisting essentially of" in certain embodiments, as desired. As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in connection with a specific amino acid or nucleotide sequence or position number, refers to the number of the specific amino acid or nucleotide sequence when compared to a given reference sequence (e.g., a reference sequence in the present invention is a (wild-type or full-length) HSD17B13 nucleic acid molecule or polypeptide). In other words, the residue (e.g., amino acid or nucleotide) number or residue (e.g., amino acid or nucleotide) position of a particular polymer is assigned based on the reference sequence, rather than by the actual position number of that residue in the specific amino acid or nucleotide sequence. For example, a particular amino acid sequence can be aligned to a reference sequence by introducing gaps to optimize residue identity between the two sequences. In these cases, even if gaps exist, the numbering of residues in the specific amino acid or nucleotide sequence is based on the reference sequence to which the sequence is aligned.

[0039] For example, nucleic acid molecules containing a thymine inserted between positions 12665 ​​and 12666 of SEQ ID NO: 1 (or position 12666 of SEQ ID NO: 2) in the HSD17B13 gene can be identified by performing sequence alignment between the specific HSD17B13 nucleic acid of interest and the nucleotide sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2. Various computational algorithms can be used to perform sequence alignment. For example, sequence alignment can be performed using the NCBI BLAST algorithm (Altschul et al., 1997, Nucleic acid molecules Res., 25, 3389-3402) or the CLUSTALW software (Sievers et al., 2014, Methods Mol. Biol., 1079, 105-116). However, sequences can also be aligned manually.

[0040] According to the present disclosure, certain alterations in HSD17B13 have been observed to be associated with a reduced risk of developing liver disease. HSD17B13 genetic variants have been identified in affected relatives that segregate with a phenotype that confers a reduced risk of developing liver disease. For example, a genetic alteration resulting in the insertion of a thymine at positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1 (or position 12666 of SEQ ID NO: 2) in the HSD17B13 gene has been observed to indicate that individuals with such an alteration may be at a reduced risk of developing liver disease. Accordingly, human subjects lacking this thymine insertion, i.e., subjects who may be at increased risk of developing or suffering from liver disease, may be treated to inhibit, alleviate, and / or suppress the onset of symptoms of liver disease. Thus, the present disclosure provides recombinant variant HSD17B13 nucleic acid molecules (including RNA or cDNA derived from RNA, or mRNA or cDNA derived from mRNA) and recombinant variant HSD17B13 polypeptides. In addition, the present disclosure provides methods that utilize the identification of such variants in a subject to identify or stratify the subject's risk for developing liver disease or to diagnose a subject as having liver disease so that at-risk subjects or subjects with active disease can be treated.

[0041] The present invention provides HSD17B13 variants that have been found to be associated with reduced levels of alanine transaminase and aspartate transaminase, a reduced risk of chronic liver disease (including nonalcoholic fatty liver disease, alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma), and a slower progression from simple fatty liver to more clinically advanced stages of chronic liver disease. The present invention also provides previously unidentified HSD17B13 gene transcripts associated with these variants.

[0042] The present invention provides nucleic acid molecules and polypeptides associated with HSD17B13 variants, and cells containing these nucleic acid molecules and polypeptides. Also provided are methods for detecting the presence of the HSD17B13 rs72613567 variant gene in a biological sample containing genomic DNA, methods for detecting the presence or level of any one of HSD17B13 transcripts C, D, E, F, F', G, and H (RNA or RNA-derived cDNA, and / or mRNA or mRNA-derived cDNA), particularly transcript D (RNA or RNA-derived cDNA, and / or mRNA or mRNA-derived cDNA), in a biological sample containing RNA or RNA-derived cDNA, mRNA or mRNA-derived cDNA, and methods for detecting the presence or level of any one of HSD17B13 proteins C, D, E, F, F', G, and H, particularly D, in a biological sample containing protein. Also provided are methods for assessing a subject's susceptibility or risk of developing liver disease. Also provided are methods for diagnosing a subject with or at risk of developing liver disease. Also provided are methods for determining a subject's risk of progressing to a clinically more advanced stage of fatty liver disease. Also provided are methods for modifying cells through the use of expression vectors to express a recombinant HSD17B13 gene or nucleic acid encoding an HSD17B13 protein.

[0043] The present disclosure provides nucleic acid molecules and polypeptides related to variants of HSD17B13 (also known as hydroxysteroid 17-beta dehydrogenase 13, 17-beta-hydroxysteroid dehydrogenase 13, 17β-hydroxysteroid dehydrogenase-13, 17β-HSD13, short-chain dehydrogenase / reductase 9, SCDR9, HMFN0376, NIIL497, and SDR16C3). The human HSD17B13 gene is approximately 19 kb in length, contains seven exons and six introns, and is located at 4q22.1 in the genome. Exemplary human HSD17B13 protein sequences have been assigned UniProt Accession No. Q7Z5P4 (Q7Z5P4-1 and Q7Z5P4-2, respectively), and NCBI Reference Sequence Nos. NP_835236 and NP_001129702. Exemplary human HSD17B13 nucleic acid molecules have been assigned NCBI Reference Sequence Nos. NM_178135 and NM_001136230.

[0044] In particular, provided is a splice variant of HSD17B13 (rs72613567) in which an adenine is inserted adjacent to the donor splice site in intron 6. This adenine is an insertion on the forward (plus) strand of the chromosome and corresponds to a thymine insertion on the reverse (minus) strand of the chromosome. Because the human HSD17B13 gene is transcribed in reverse, this nucleotide insertion is reflected as a thymine insertion in the exemplary variant HSD17B13 rs72613567 sequence shown in SEQ ID NO:2 relative to the exemplary wild-type HSD17B13 gene sequence shown in SEQ ID NO:1. Thus, this insertion is referred to as a thymine inserted between positions 12665 ​​and 12666 in SEQ ID NO:1 or at position 12666 in SEQ ID NO:2.

[0045] Two transcripts (transcript A (SEQ ID NO: 21) and transcript B (SEQ ID NO: 22)) were previously identified to be expressed in subjects with a wild-type HSD17B13 gene. Transcript A contains all seven exons of the HSD17B13 gene, while transcript B skips exon 2. Transcript A is the dominant transcript in wild-type subjects. However, the present invention provides six additional, previously unidentified, expressed HSD17B13 transcripts: transcript C of SEQ ID NO: 23, transcript D of SEQ ID NO: 24, transcript E of SEQ ID NO: 25, transcript F of SEQ ID NO: 26, transcript G of SEQ ID NO: 28, and transcript H of SEQ ID NO: 29. Transcript C skips exon 6 compared to transcript A. Transcript D, compared to transcript A, has a guanine insertion 3' of exon 6, resulting in a frameshift at exon 7 and premature truncation of exon 7. Transcript E, compared to transcript A, has an additional exon between exons 3 and 4. Transcript F, which is expressed only in HSD17B13 rs72613567 variant carriers, shows a read-through from exon 6 to intron 6 compared to transcript A. Transcript G, compared to transcript A, skips exon 2 and has a guanine insertion 3' of exon 6, resulting in a frameshift at exon 7 and premature truncation of exon 7. Transcript H, compared to transcript A, has an additional exon between exons 3 and 4, resulting in a guanine insertion 3' of exon 6, resulting in a frameshift at exon 7 and premature truncation of exon 7. In carriers of the HSD17B13 rs72613567 variant, transcripts C, D, F, G, and H are predominant, with transcript D being the most abundant transcript in carriers of the HSD17B13 rs72613567 variant. Also provided herein is one additional, previously unidentified HSD17B13 transcript (F', SEQ ID NO: 27) that is expressed at low levels.Like transcript F, transcript F' exhibits a read-through region from exon 6 to intron 6 compared to transcript A, but in contrast to transcript F, this read-through region does not contain the thymine insertion present in the HSD17B13 rs72613567 variant gene.

[0046] 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 proceeding from the 5' to the 3' end of the sequence (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, although the complementary strand should be understood by reference to the displayed strand in each case. The amino acid sequences follow the standard convention of proceeding from the amino to the carboxy terminus of the sequence (i.e., from left to right in each sequence).

[0047] SEQ ID NO: 1 is the wild-type HSD17B13 genomic sequence (Human Genome Assembly GRCh38). Common transcripts found in subjects with the wild-type HSD17B13 gene include transcript A, transcript B, transcript E, and transcript F'.

[0048] SEQ ID NO: 2 is the HSD17B13 genomic sequence variant (Human Genome Assembly GRCh38, rs72613567, T insertion at chr4:87310241-87310240, T insertion at position 12666). Common transcripts found in subjects with the variant rs72613567 HSD17B13 gene include transcript C, transcript D, transcript F, transcript G, and transcript H.

[0049] SEQ ID NO: 53 is the endogenous HSD17B13 promoter (499-100 upstream of the transcription start site (TSS)). As used herein, the term "transcript" means any one or more of the RNA or mRNA molecules disclosed in the table below, or the corresponding cDNA molecules derived therefrom, unless otherwise indicated by the context in which the term is used. The sequence identifier names for the various transcripts are listed in the table below. RNA transcripts are shown along with their cDNA counterparts, and mRNA transcripts are shown along with their cDNA counterparts.

[0050] [Table 1]

[0051] Thus, as used herein, unless the context indicates otherwise, the term "transcript A" means any one or more of SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:21 and / or SEQ ID NO:30; "transcript B" means any one or more of SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:22 and / or SEQ ID NO:31; "transcript C" means any one or more of SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:23 and / or SEQ ID NO:32; "transcript D" means i) any one or more of SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24 and / or SEQ ID NO:33; and "transcript E" means i) any one or more of SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24 and / or SEQ ID NO:33. , SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:25 and / or SEQ ID NO:34; "transcript F" means one or more of SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26 and / or SEQ ID NO:35; "transcript F'" means one or more of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27 and / or SEQ ID NO:36; "transcript G" means one or more of SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:28 and / or SEQ ID NO:37; and "transcript H" means one or more of SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:29 and / or SEQ ID NO:38.

[0052] For each transcript, the nucleotide positions of the exons in the HSD17B13 gene are shown below. Nucleotide positions in SEQ ID NO: 1 of exons of HSD17B13 transcripts commonly found in subjects homozygous for the wild-type HSD17B13 gene

[0053] [Table 2]

[0054] *Includes the read-through region from exon 6 to intron 6. Read-through region = positions 12665 ​​to 13501 rs72613567 Nucleotide position in SEQ ID NO: 2 of the exon of the HSD17B13 transcript commonly found in subjects homozygous for the HSD17B13 variant gene (insertion of T at position 12666)

[0055] [Table 3]

[0056] ∧Compared to transcript A, it contains an additional residue 12665 ​​at the 3' end. *Includes the read-through region from exon 6 to intron 6. Read-through region = 12665-13502 The corresponding HSD17B13 isoform proteins include: i) isoform A (SEQ ID NO: 39; region encoded by exon 1 = 1-70, region encoded by exon 2 = 71-106, region encoded by exon 3 = 107-150, region encoded by exon 4 = 151-185, region encoded by exon 5 = 186-232, region encoded by exon 6v1 = 233-271, region encoded by exon 7 = 272-300); ii) protein isoform B (SEQ ID NO: 40 , the region encoded by exon 1 = 1-70, exon 2 = skipped, the region encoded by exon 3 = 71-114, the region encoded by exon 4 = 115-149, the region encoded by exon 5 = 150-196, the region encoded by exon 6v1 = 197-235, the region encoded by exon 7 = 236-264), iii) protein isoform C (SEQ ID NO: 41, the region encoded by exon 1 = 1-70, the region encoded by exon 2 = 71-106, exon 3 = 107-150, region encoded by exon 4 = 151-185, region encoded by exon 5 = 186-232, exon 6 = skipped, region encoded by exon 7 = 233-261), iv) protein isoform D (SEQ ID NO: 42, region encoded by exon 1 = 1-70, region encoded by exon 2 = 71-106, region encoded by exon 3 = 107-150, region encoded by exon 4 = 151-185, region encoded by exon 5 = 233-261), region encoded by exon 6v1 = 186-232, region encoded by exon 6v2 = 233-271, region encoded by exon 7 = 272-274), v) protein isoform E (SEQ ID NO: 43, region encoded by exon 1 = 1-70, region encoded by exon 2 = 71-106, region encoded by exon 3 = 107-150, region encoded by exon 3' = 151-174, region encoded by exon 4 = 175-209, region encoded by exon 5 = 210-256,vi) protein isoform F (SEQ ID NO: 44; region encoded by exon 1 = 1-70; region encoded by exon 2 = 71-106; region encoded by exon 3 = 107-150; region encoded by exon 4 = 151-185; region encoded by exon 5 = 186-232; region encoded by exon 6v3 = 186-232; vii) protein isoform F' (SEQ ID NO: 45; region encoded by exon 1 = 1-70; region encoded by exon 2 = 71-106; region encoded by exon 3 = 107-150; region encoded by exon 4 = 151-185; region encoded by exon 5 = 186-232; region encoded by exon 6v4 = 23 3-271), viii) protein isoform G (SEQ ID NO: 46, region encoded by exon 1 = 1-70, exon 2 = skipped, region encoded by exon 3 = 71-114, region encoded by exon 4 = 115-149, region encoded by exon 5 = 150-196, region encoded by exon 6v2 = 197-235, region encoded by exon 7 = 236-238), and ix) protein isoform H (SEQ ID NO: 47, region encoded by exon 1 = 1 to 70, region encoded by exon 2 = 71 to 106, region encoded by exon 3 = 107 to 150, region encoded by exon 3' = 151 to 174, region encoded by exon 4 = 175 to 209, region encoded by exon 5 = 210 to 256, region encoded by exon 6v2 = 257 to 295, region encoded by exon 7 = 296 to 298).

[0057] As described in more detail elsewhere herein, the HSD17B13 rs72613567 variant is associated with reduced levels of alanine transaminase and aspartate transaminase and a reduced risk of chronic liver disease (including nonalcoholic fatty liver disease, alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma). The HSD17B13 rs72613567 variant is also associated with a reduced progression from simple fatty liver to more clinically advanced stages of chronic liver disease.

[0058] Disclosed herein are variant HSD17B13 nucleic acid molecules (including variant HSD17B13 genes and variant HSD17B13 transcripts). Also disclosed are nucleic acid molecules that hybridize under stringent or moderate conditions to any of the nucleic acid molecules disclosed herein. Such nucleic acid molecules can be useful, for example, to express HSD17B13 variant proteins or as primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in further detail elsewhere herein. In any of the embodiments described herein, the nucleic acid molecules and / or polypeptides of the present disclosure can be isolated nucleic acid molecules or isolated polypeptides.

[0059] The present disclosure provides a nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides of the HSD17B13 gene, which contiguous nucleotides are at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequence in SEQ ID NO: 2, and which has a thymine at a position corresponding to position 12666 of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule of the present disclosure comprises or consists of at least 15 contiguous nucleotides of the HSD17B13 gene, which contiguous nucleotides are at least about 90% identical to the corresponding sequence in SEQ ID NO: 2, and which has a thymine at a position corresponding to position 12666 of SEQ ID NO: 2.

[0060] The present disclosure provides a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding a polypeptide having an amino acid sequence 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 the amino acid sequence of HSD17B13 isoform D (SEQ ID NO:42). In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding a polypeptide having an amino acid sequence at least about 90% identical to the amino acid sequence of HSD17B13 isoform D (SEQ ID NO:42). In some embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence encoding a polypeptide having the amino acid sequence of HSD17B13 isoform D (SEQ ID NO:42). In some embodiments, these nucleic acid molecules encode a polypeptide having 274 amino acids. In some embodiments, these nucleic acid molecules encode a polypeptide having a C-terminal Val-Ser-Ser. In some embodiments, these nucleic acid molecules encode polypeptides that are associated with a reduced risk of developing any of the liver diseases described herein or a reduced risk of progressing to a more clinically advanced stage of fatty liver disease.

[0061] The present disclosure provides a nucleic acid molecule comprising at least 15 contiguous nucleotides of the HSD17B13 gene and having a thymine at a position corresponding to position 12666 of the HSD17B13 rs72613567 variant gene (SEQ ID NO: 2) (or having a thymine at positions 12666 and 12667). That is, disclosed herein is a nucleic acid molecule comprising at least 15 contiguous nucleotides of the HSD17B13 gene and having a thymine inserted between the nucleotides corresponding to positions 12665 ​​and 12666 of the wild-type HSD17B13 gene (SEQ ID NO: 1). Such nucleic acid molecules can be useful, for example, for expressing HSD17B13 variant transcripts and isoform proteins.

[0062] The HSD17B13 gene can be an HSD17B13 gene obtained from any organism. For example, the HSD17B13 gene can be a human HSD17B13 gene or an ortholog from another organism (such as a non-human mammal, a rodent, a mouse, or a rat). It is known that gene sequences within a population can vary due to polymorphisms (such as single nucleotide polymorphisms). The examples shown herein are merely illustrative sequences. Other sequences are also possible. For example, the at least 15 consecutive nucleotides described above can be at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequence in the HSD17B13 rs72613567 variant (SEQ ID NO: 2) (including positions 12666 or 12666 and 12667 of SEQ ID NO: 2). In some embodiments, the at least 15 contiguous nucleotides can be at least about 90% identical to the corresponding sequence in the HSD17B13 rs72613567 variant (SEQ ID NO: 2) (including positions 12666, or 12666 and 12667 of SEQ ID NO: 2). In some embodiments, a nucleic acid molecule of the present disclosure comprises at least 15 contiguous nucleotides of SEQ ID NO: 2, including positions 12666, or 12666 and 12667 of SEQ ID NO: 2. In some embodiments, the at least 15 contiguous nucleotides can be at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequence in the wild-type HSD17B13 gene (SEQ ID NO: 1) (including positions 12665 ​​and 12666 of SEQ ID NO: 1), wherein a thymine is present between positions 12665 ​​and 12666 of SEQ ID NO: 1. In some embodiments, the at least 15 consecutive nucleotides can be at least about 90% identical to the corresponding sequence in the wild-type HSD17B13 gene (SEQ ID NO: 1) (including positions 12665 ​​and 12666 of SEQ ID NO: 1), and a thymine is present between positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1.In some embodiments, a nucleic acid molecule of the present disclosure comprises at least 15 contiguous nucleotides of SEQ ID NO:1, including positions 12665 ​​and 12666 of SEQ ID NO:1, and a thymine is present between positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1.

[0063] In some cases, an isolated nucleic acid molecule of the invention can comprise an HSD17B13 minigene in which one or more non-essential segments of the gene are deleted compared to the corresponding wild-type HSD17B13 gene. By way of example, the deleted segments include one or more intron sequences. In some embodiments, the HSD17B13 minigene can comprise, for example, exons corresponding to exons 1-7 of HSD17B13 transcript D and an intron corresponding to intron 6 in SEQ ID NO:2. In some embodiments, the HSD17B13 minigene can comprise exons 1-7 and intron 6 of SEQ ID NO:2. Minigenes are described in further detail elsewhere herein.

[0064] The present disclosure provides nucleic acid molecules corresponding to all or a portion of an RNA transcript (such as transcript A, transcript B, transcript C, transcript D, transcript E, transcript F, transcript F', transcript G, and transcript H) or their corresponding cDNAs, or an mRNA transcript (such as transcript A, transcript B, transcript C, transcript D, transcript E, transcript F, transcript F', transcript G, and transcript H) or their corresponding cDNAs.

[0065] The present disclosure provides nucleic acid molecules corresponding to all or a portion of the RNA transcripts (such as Transcript C, Transcript D, Transcript E, Transcript F, Transcript F', Transcript G, and Transcript H) or their corresponding cDNAs, or the mRNA transcripts (such as Transcript C, Transcript D, Transcript E, Transcript F, Transcript F', Transcript G, and Transcript H) or their corresponding cDNAs.

[0066] The present disclosure provides nucleic acid molecules corresponding to all or a portion of the RNA transcripts (such as transcript C, transcript D, transcript F, transcript G, and transcript H) or their corresponding cDNAs, or the mRNA transcripts (such as transcript C, transcript D, transcript F, transcript G, and transcript H) or their corresponding cDNAs.

[0067] The present disclosure provides RNA transcript D or the corresponding cDNA, or a nucleic acid molecule corresponding to all or a portion of mRNA transcript D or the corresponding cDNA. Such isolated nucleic acid molecules can be useful, for example, to express HSD17B13 variant transcripts and proteins.

[0068] In some embodiments, a nucleic acid molecule of the disclosure comprises or consists of a nucleotide sequence that is 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 the nucleotide sequence of HSD17B13 transcript D (SEQ ID NO: 6, 15, 24, or 33). In some embodiments, a nucleic acid molecule of the disclosure is RNA and its cDNA comprises or consists of SEQ ID NO: 6, or comprises or consists of SEQ ID NO: 24, or the nucleic acid molecule is mRNA and its cDNA comprises or consists of SEQ ID NO: 15, or comprises or consists of SEQ ID NO: 33.

[0069] Compared to transcript A, HSD17B13 transcripts D, G, and H contain a guanine insertion at the 3' end of exon 6, resulting in a frameshift in exon 7 and premature truncation of the region of HSD17B13 protein encoded by exon 7. Thus, the present invention provides nucleic acid molecules that contain a segment (e.g., at least 15 contiguous nucleotides) present in transcripts D, G, and H (or fragments or homologs thereof) that is absent in transcript A (or fragments or homologs thereof). The present invention also provides nucleic acid molecules that contain a segment (e.g., at least 15 contiguous nucleotides) present in transcript D (or fragments or homologs thereof) that is absent in transcript A (or fragments or homologs thereof). Such regions can be readily identified by comparing the sequences of the transcripts.For example, the present invention provides a nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) encoding all or a portion of an HSD17B13 protein, wherein a segment of the contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region spanning the boundary between exons 6 and 7 of transcript D, transcript G, or transcript H, and wherein the segment is The nucleic acid molecule is a nucleic acid molecule in which the transcript contains a guanine at the residue corresponding to residue 878 at the 3' end of exon 6 of transcript D (i.e., in addition to the guanine at the start of exon 7, a guanine is inserted at the 3' end of exon 6 compared to transcript A), a guanine at the residue corresponding to residue 770 at the 3' end of exon 6 of transcript G (i.e., in addition to the guanine at the start of exon 7, a guanine is inserted at the 3' end of exon 6 compared to transcript B), or a guanine at the residue corresponding to residue 950 at the 3' end of exon 6 of transcript H (i.e., in addition to the guanine at the start of exon 7, a guanine is inserted at the 3' end of exon 6 compared to transcript E). It is understood that such nucleic acids will contain a sufficient number of nucleotides in each of exon 6 and exon 7 to distinguish the inserted guanine from other features in the HSD17B13 transcript (e.g., the guanine at the start of exon 7, the read-through into intron 6 in transcript F, or the deleted exon 6 in transcript C).

[0070] By way of example, a nucleic acid molecule of the present disclosure can comprise or consist of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) of transcript D, spanning the boundary between exons 6 and 7, optionally including exons 6 and 7 of transcript D, and optionally including the entire sequence of transcript D.

[0071] In some embodiments, a nucleic acid molecule of the present disclosure further comprises a segment present in transcript D (or a fragment or homolog thereof) that is absent in transcript G (or a fragment or homolog thereof), and the nucleic acid molecule further comprises a segment present in transcript D (or a fragment or homolog thereof) that is absent in transcript H (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the transcripts. For example, such a nucleic acid molecule can comprise or consist of a segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the region spanning the boundary between exon 3 and exon 4 of transcript D, in order to distinguish it from transcript H. Similarly, such nucleic acid molecules can comprise or consist of a segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region within exon 2 of transcript D, a region spanning the boundary between exons 1 and 2 of transcript D, or a region spanning the boundary between exons 2 and 3 of transcript D, in order to distinguish it from transcript G. In some embodiments, a nucleic acid molecule of the present disclosure can comprise or consist of a sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence set forth in transcript D and encodes an HSD17B13 isoform protein comprising the sequence set forth in isoform D. Like transcript D, transcript H contains a guanine insertion 3' of exon 6 compared to transcript A.Transcript H further contains an additional exon (exon 3') between exon 3 and exon 4 compared to transcripts A and D. Thus, the present invention provides nucleic acid molecules as described above that include segments present in transcripts D, G, and H (or fragments or homologs thereof) that are absent in transcript A (or fragments or homologs), but further include a segment of transcript H (or fragments or homologs) that is absent in transcript D (or fragments or homologs) (e.g., at least 15 contiguous nucleotides). Such regions can be readily identified by comparing the sequences of the transcripts. For example, the present invention provides a nucleic acid molecule as described with respect to transcript D, wherein a segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) thereof is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region within exon 3' of transcript H, a region spanning the boundary between exons 3 and 3' of transcript H, or a region spanning the boundary between exons 3' and 4 of transcript H. It is understood that such a nucleic acid molecule will include a sufficient number of nucleotides in each of exon 3 and exon 3', or each of exon 3' and exon 4, to be distinct from other features in the HSD17B13 transcript (e.g., the boundary between exons 3 and 4). For example, the region of exon 3' can include the entirety of exon 3'. Optionally, the nucleic acid molecules of the present disclosure can comprise or consist of a sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence shown in transcript H, and encode an HSD17B13 protein, including isoform H.

[0072] By way of example, the nucleic acid molecule can comprise or consist of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) of transcript H, including a region within exon 3', a region spanning the exon 3-exon 3' boundary, or a region spanning the exon 3'-exon 4 boundary, optionally including the entire exon 3' of transcript H, and optionally including the entire sequence of transcript H.

[0073] Like transcript D, transcript G contains a guanine insertion 3' of exon 6 compared to transcript A. However, in addition, transcript G lacks exon 2 compared to transcripts A and D (i.e., transcript G contains the boundary between exons 1 and 3, which is not present in transcripts A and D). Thus, the present invention provides nucleic acid molecules as described above that comprise or consist of a segment present in transcripts D, G, and H (or fragments or homologs thereof) that is not present in transcript A (or fragments or homologs), but that further contain a segment (e.g., at least 15 contiguous nucleotides) from transcript G (or fragments or homologs) that is not present in transcript D (or fragments or homologs). Such regions can be readily identified by comparing the sequences of the transcripts. For example, the invention provides nucleic acid molecules described with respect to transcript D, wherein a segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) thereof is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region spanning the boundary of exon 1 and exon 3 of transcript G. It is understood that such nucleic acid molecules will include a sufficient number of nucleotides in each of exon 1 and exon 3 to be distinct from other features in the HSD17B13 transcript (e.g., the boundary between exon 1 and exon 2 or the boundary between exon 2 and exon 3). For example, the region can include the entirety of exon 1 and exon 3 of transcript G. Optionally, a nucleic acid molecule of the present disclosure comprises or consists of a sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence shown in transcript G, and encodes an HSD17B13 protein that comprises the sequence shown in isoform G.

[0074] By way of example, a nucleic acid molecule of the present disclosure can comprise or consist of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) of transcript G, including the region spanning the boundary between exon 1 and exon 3, optionally including exon 1 and exon 3 of transcript G, and optionally including the entire sequence of transcript G.

[0075] Also provided herein are nucleic acid molecules that comprise or consist of a segment (e.g., at least 15 contiguous nucleotides) present in transcript E (or a fragment or homolog thereof) that is absent in transcript A (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript E contains an additional exon between exon 3 and exon 4. Thus, the present invention provides a nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) encoding all or a portion of an HSD17B13 protein, wherein the segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region within exon 3' of transcript E, a region spanning the boundary between exons 3 and 3' of transcript E, or a region spanning the boundary between exons 3' and 4 of transcript E. It is understood that such a nucleic acid molecule will comprise a sufficient number of nucleotides in each of exon 3 and exon 3', or each of exon 3' and exon 4, to be distinct from other features in the HSD17B13 transcript (e.g., the boundary between exons 3 and 4). For example, the region of exon 3' can include the entire exon 3'. Optionally, a nucleic acid molecule of the present disclosure further includes a segment (e.g., at least 15 contiguous nucleotides) from transcript E (or a fragment or homolog thereof) that is not present in transcript H (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the transcripts.For example, the invention provides a nucleic acid molecule as described above, wherein a segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) thereof is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region spanning the boundary between exons 6 and 7 of transcript E. It is understood that such a nucleic acid molecule will include a sufficient number of nucleotides in each of exons 6 and 7 to distinguish it from other features in the HSD17B13 transcript (particularly the additional guanine at the 3' end of exon 6 of transcript H). For example, the region can include the entirety of exons 6 and 7 of transcript E. Optionally, the isolated nucleic acid of the present disclosure comprises or consists of a sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence set forth in transcript E, and encodes an HSD17B13 protein comprising the sequence set forth in isoform E.

[0076] By way of example, a nucleic acid molecule of the present disclosure can comprise or consist of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) of transcript E, including a region within exon 3', a region spanning the boundary between exon 3 and exon 3', or a region spanning the boundary between exon 3' and exon 4, optionally including the entire exon 3' of transcript E, and optionally including the entire sequence of transcript E.

[0077] Also provided herein are nucleic acid molecules comprising or consisting of a segment (e.g., at least 15 contiguous nucleotides) present in transcript F (or a fragment or homolog thereof) that is not present in transcript A (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript F contains a read-through region from exon 6 to intron 6, which read-through region contains a thymine insertion present in the HSD17B13 rs72613567 variant gene. Thus, the present invention provides nucleic acid molecules comprising or consisting of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) encoding all or a portion of an HSD17B13 protein, wherein the segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region within the readthrough into intron 6 in transcript F or to a region spanning the boundary between the readthrough into intron 6 and the remainder of exon 6 in transcript F. It is understood that such nucleic acid molecules will contain a sufficient number of nucleotides in the readthrough portion to distinguish the readthrough portion from other features in the HSD17B13 transcript (e.g., the boundary between exons 6 and 7 in other HSD17B13 transcripts). Optionally, the contiguous nucleotides include a sequence present in transcript F that is not present in transcript F' (i.e., a thymine insertion). Transcript F' also includes a readthrough region from exon 6 to intron 6 compared to transcript A, but the readthrough region does not include the thymine insertion present in the HSD17B13 rs72613567 variant gene. For example, the region can be the entire readthrough region into intron 6 in transcript F.Optionally, an isolated nucleic acid molecule of the present disclosure comprises or consists of a sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence set forth in transcript F, and encodes an HSD17B13 protein comprising the sequence set forth in protein isoform F.

[0078] By way of example, a nucleic acid molecule of the present disclosure can comprise or consist of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) of transcript F, including a region within the read-through into intron 6 or spanning the boundary between the read-through into intron 6 and the remainder of exon 6, optionally including the entire read-through into intron 6, and optionally including the entire sequence of transcript F.

[0079] The present invention also provides a nucleic acid molecule comprising or consisting of a segment (e.g., at least 15 contiguous nucleotides) present in transcript F' (or a fragment or homolog thereof) that is not present in transcript A (or a fragment or homolog thereof). Such a region can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript F' contains a read-through region from exon 6 to intron 6, which does not contain the thymine insertion present in the HSD17B13 rs72613567 variant gene. Thus, the present invention provides nucleic acid molecules comprising or consisting of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) encoding all or a portion of an HSD17B13 protein, wherein the segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a region within the readthrough into intron 6 in transcript F' or to a region spanning the boundary between the readthrough into intron 6 and the remainder of exon 6 in transcript F'. It is understood that such nucleic acid molecules will contain a sufficient number of nucleotides in the readthrough portion to distinguish the readthrough portion from other features in the HSD17B13 transcript (e.g., the boundary between exons 6 and 7 in other HSD17B13 transcripts). Optionally, the contiguous nucleotides include a sequence present in transcript F' that is not present in transcript F. The readthrough region in transcript F includes a thymine insertion present in the HSD17B13 rs72613567 variant gene, and the thymine insertion is not included in the readthrough region in transcript F'. For example, the region can be the entire readthrough region into intron 6 in transcript F'.Optionally, an isolated nucleic acid molecule of the present disclosure comprises or consists of a sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence set forth in transcript F', and encodes an HSD17B13 protein that comprises, consists essentially of, or consists of the sequence set forth in isoform F'.

[0080] By way of example, a nucleic acid molecule of the present disclosure can comprise or consist of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) of transcript F', including a region within the read-through into intron 6 or spanning the boundary between the read-through into intron 6 and the remainder of exon 6, optionally including the entire read-through into intron 6, and optionally including the entire sequence of transcript F'.

[0081] Also provided herein are nucleic acid molecules that comprise or consist of a segment (e.g., at least 15 contiguous nucleotides) present in transcript C (or a fragment or homolog thereof) that is not present in transcript A (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript C lacks exon 6 (i.e., transcript C includes the boundary between exons 5 and 7, which is not present in transcript A). Thus, the present invention provides nucleic acid molecules comprising or consisting of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) encoding all or a portion of an HSD17B13 protein, wherein the segment of contiguous nucleotides (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the region spanning the boundary between exons 5 and 7 of transcript C. It is understood that such nucleic acid molecules will include a sufficient number of nucleotides in each of exon 5 and exon 7 to be distinct from other features in the HSD17B13 transcript (e.g., the boundary between exons 5 and 6 or the boundary between exons 6 and 7 of other HSD17B13 transcripts). For example, the region can include the entirety of exons 5 and 7 of transcript C. Optionally, the nucleic acid molecule of the invention comprises or consists of a sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the sequence shown in transcript C, and encodes an HSD17B13 protein comprising the sequence shown in isoform C.

[0082] By way of example, a nucleic acid molecule of the present disclosure can comprise or consist of at least 15 contiguous nucleotides (e.g., at least 20 contiguous nucleotides or at least 30 contiguous nucleotides) of transcript C, including the region spanning the boundary between exon 5 and exon 7, optionally including the entirety of exon 5 and exon 7 of transcript C, and optionally including the entire sequence of transcript C.

[0083] In some embodiments, nucleic acid molecules of the present disclosure comprise fewer nucleotides than the entire sequence of an HSD17B13 transcript. In some embodiments, nucleic acid molecules of the present disclosure comprise or consist of at least about 5, at least about 8, at least about 10, at least about 12, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 600 contiguous nucleotides of a particular transcript. In some embodiments, nucleic acid molecules of the present disclosure comprise or consist of at least about 200 to at least about 500 contiguous nucleotides of a particular transcript. In this regard, longer nucleic acid molecules are preferred over shorter nucleic acid molecules. In some embodiments, nucleic acid molecules of the present disclosure comprise or consist of at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, or at least about 500 contiguous nucleotides of a particular transcript, with longer nucleic acid molecules being preferred over shorter ones in this regard.

[0084] In some embodiments, the nucleic acid molecules of the present disclosure can be useful, for example, as primers and probes. The present disclosure provides a nucleic acid molecule comprising or consisting of about 5 nucleotides up to about 50 nucleotides that specifically hybridizes to the HSD17B13 gene or its complement in a region including a position corresponding to position 12666 of SEQ ID NO:2, wherein the nucleic acid molecule has a thymine at the position corresponding to position 12666 of SEQ ID NO:2 or its complement.

[0085] The present disclosure provides a nucleic acid molecule comprising or consisting of from about 5 up to about 50 nucleotides that specifically hybridizes to variant HSD17B13 transcript D, wherein the nucleic acid molecule specifically hybridizes to i) a nucleotide sequence that is 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 the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or ii) the complement of the nucleotide sequence in i). In some embodiments, the nucleic acid molecule of the present disclosure comprises or consists of from about 5 up to about 50 nucleotides that specifically hybridizes to variant HSD17B13 transcript D, wherein the nucleic acid molecule specifically hybridizes to i) a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or ii) the complement of the nucleotide sequence in i).

[0086] The present disclosure provides nucleic acid molecules comprising or consisting of from about 5 nucleotides up to about 50 nucleotides comprising, or consisting of: i) a nucleotide sequence that is 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 (i.e., at least about 90% identical) to the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or its complement; ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA); and / or iii) a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA). In some embodiments, a nucleic acid molecule of the disclosure specifically hybridizes to an RNA molecule comprising or consisting of SEQ ID NO:6, or its cDNA comprising or consisting of SEQ ID NO:24, or the nucleic acid molecule specifically hybridizes to an mRNA comprising or consisting of SEQ ID NO:15, its cDNA comprising or consisting of SEQ ID NO:33, or their complements. In some embodiments, a nucleic acid molecule of the disclosure is linked to a heterologous nucleic acid or comprises a heterologous label.

[0087] In some embodiments, such nucleic acid molecules are 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, The nucleic acid molecule of the present disclosure comprises or consists of 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, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, at least about 10000, at least about 11000, or at least about 11500 nucleotides. In some embodiments, the nucleic acid molecule of the present disclosure comprises or consists of at least 15 nucleotides. In some embodiments, the nucleic acid molecule of the present disclosure comprises or consists of at least 15 nucleotides to at least about 35 nucleotides. In some embodiments, such nucleic acid molecules hybridize under stringent conditions to variant HSD17B13 genomic DNA, variant HSD17B13 minigene, variant HSD17B13 RNA (or cDNA derived from RNA), or variant HSD17B13 mRNA (or cDNA derived from mRNA). Such nucleic acid molecules may be used, for example, as probes, primers, variation-specific probes, or variation-specific primers, as described or exemplified herein.

[0088] Also disclosed herein is a nucleic acid molecule comprising or consisting of at least 15 consecutive nucleotides that hybridize to an HSD17B13 gene (e.g., an HSD17B13 minigene) in a segment including, or within 1000, 500, 400, 300, 200, 100, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 nucleotides of, position 12666 or positions 12666 and 12667 of the HSD17B13 rs72613567 variant (SEQ ID NO: 2). Such nucleic acid molecules can be useful, for example, as primers or probes.

[0089] In some embodiments, the at least 15 contiguous nucleotides can hybridize to a segment of the HSD17B13 gene or HSD17B13 minigene that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical (or at least about 90% identical) to the corresponding sequence in the HSD17B13 rs72613567 variant (SEQ ID NO: 2) and that has a thymine at a position corresponding to position 12666 of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule of the present disclosure can hybridize to at least 15 contiguous nucleotides of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule of the present disclosure hybridizes to a segment including positions 12666 or 12666 and 12667 in SEQ ID NO: 2, or positions 12666 or 12666 and 12667 in SEQ ID NO: 2.

[0090] In some embodiments, a segment to which a nucleic acid molecule of the present disclosure can hybridize can comprise, for example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 75, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000 consecutive nucleotides of a nucleic acid molecule encoding a variant HSD17B13 protein isoform. In some embodiments, the segment to which a nucleic acid molecule of the disclosure can hybridize can be, for example, at most 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 75, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides of a nucleic acid encoding a variant HSD17B13 protein isoform. In some embodiments, a nucleic acid molecule of the disclosure can include, for example, at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, or 19000 contiguous nucleotides of a variant HSD17B13 gene. In some embodiments, a segment to which a nucleic acid molecule of the disclosure can hybridize can be, for example, at most 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 75, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides of a variant HSD17B13 gene. In some embodiments, the segment can be about 15-100 nucleotides in length or about 15-35 nucleotides in length.

[0091] Also provided are nucleic acid molecules that hybridize to segments of an RNA transcript (such as Transcript A, Transcript B, Transcript C, Transcript D, Transcript E, Transcript F, Transcript F', Transcript G, and Transcript H) or their corresponding cDNAs, or an mRNA transcript (such as Transcript A, Transcript B, Transcript C, Transcript D, Transcript E, Transcript F, Transcript F', Transcript G, and Transcript H) or their corresponding cDNAs.

[0092] Also provided are nucleic acid molecules that hybridize to segments of the RNA transcripts (such as Transcript C, Transcript D, Transcript E, Transcript F, Transcript F', Transcript G, and Transcript H) or their corresponding cDNAs, or to segments of the mRNA transcripts (such as Transcript C, Transcript D, Transcript E, Transcript F, Transcript F', Transcript G, and Transcript H) or their corresponding cDNAs.

[0093] Also provided are nucleic acid molecules that hybridize to segments of the RNA transcripts (such as Transcript C, Transcript D, Transcript F, Transcript G, and Transcript H) or their corresponding cDNAs, or to segments of the mRNA transcripts (such as Transcript C, Transcript D, Transcript F, Transcript G, and Transcript H) or their corresponding cDNAs.

[0094] Also provided are nucleic acid molecules that hybridize to RNA transcript D or the corresponding cDNA, or to a segment of mRNA transcript D or the corresponding cDNA. The present invention provides nucleic acid molecules that contain or consist of a region (e.g., at least 15 contiguous nucleotides) that hybridizes to a segment present in transcripts D, G, and H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, but not in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof. Such regions can be readily identified by comparing the sequences of the transcripts. For example, the present invention provides a nucleic acid molecule that hybridizes to at least 15 contiguous nucleotides of a nucleic acid encoding an HSD17B13 protein, wherein the contiguous nucleotides are 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 (i.e., at least about 90% identical) to a region spanning the boundary between exon 6 and exon 7 of transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA). A nucleic acid molecule comprising or consisting of a segment (e.g., at least 5 consecutive nucleotides, at least 10 consecutive nucleotides, or at least 15 consecutive nucleotides) of exon 6 of transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or RNA), which segment contains a guanine at the residue corresponding to residue 878 at the 3' end of exon 6 (i.e., in addition to the guanine at the start of exon 7, a guanine is inserted at the 3' end of exon 6 compared to transcript A).Alternatively, the present invention provides a nucleic acid molecule that hybridizes to at least 15 contiguous nucleotides of a segment of a nucleic acid encoding HSD17B13 protein, wherein the contiguous nucleotides comprise or consist of a segment (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) that is 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 (i.e., at least about 90% identical) to a region spanning the boundary between exons 6 and 7 of transcript G, and the segment contains a guanine at the residue corresponding to residue 770 at the 3' end of exon 6 of transcript G (RNA or RNA-derived cDNA, and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA) (i.e., in addition to the guanine at the start of exon 7, a guanine is inserted at the 3' end of exon 6 compared to transcript B). Alternatively, the present invention provides a nucleic acid molecule that hybridizes to at least 15 contiguous nucleotides of a nucleic acid encoding an HSD17B13 protein, wherein the contiguous nucleotides comprise or consist of a segment (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) that is 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 (i.e., at least about 90% identical) to a region spanning the boundary between exon 6 and exon 7 of transcript H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or RNA), and the segment includes a guanine at the residue corresponding to residue 950 at the 3' end of exon 6 of transcript H (i.e., in addition to the guanine at the start of exon 7, a guanine is inserted at the 3' end of exon 6 compared to transcript E).It is understood that such nucleic acid molecules would be designed to hybridize to a sufficient number of nucleotides in each of exon 6 and exon 7 to distinguish the inserted guanine from other features in the HSD17B13 transcript (e.g., the read-through into intron 6 in transcript F or the deletion of exon 6 in transcript C).

[0095] As one example, the segment can comprise or consist of the region of transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) spanning the boundary between exons 6 and 7 (i.e., the region including the guanine at residue 878 of transcript D). As another example, the segment can comprise or consist of the region of transcript G (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) spanning the boundary between exons 6 and 7 (i.e., the region including the guanine at residue 770 of transcript G). As another example, the segment can include or consist of the region of transcript H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA) spanning the boundary between exons 6 and 7 (i.e., the region including the guanine at residue 950 of transcript H).

[0096] In some embodiments, the nucleic acid molecule of the present disclosure further comprises or consists of a region (e.g., 15 contiguous nucleotides) that hybridizes to a segment present in transcript D (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, that is not present in transcript G (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof; the nucleic acid molecule further comprises or consists of a region that hybridizes to a segment present in transcript D (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, that is not present in transcript H (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof. Such segments can be readily identified by comparing the sequences of the transcripts.For example, a segment (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) present in transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, that is not present in transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, can be at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical (i.e., at least about 90% identical) to a region spanning the boundary between exon 3 and exon 4 of transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in order to be distinguished from transcript H.Similarly, a segment (e.g., at least 5 consecutive nucleotides, at least 10 consecutive nucleotides, or at least 15 consecutive nucleotides) present in transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, that is not present in transcript G (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, is referred to as a segment (e.g., at least 5 consecutive nucleotides, at least 10 consecutive nucleotides, or at least 15 consecutive nucleotides) present in transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, in order to distinguish it from transcript G. The transcript D may be at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical (i.e., at least about 90% identical) to a region within exon 2 of a cDNA derived from transcript A, preferably an RNA or RNA-derived cDNA), a region spanning the boundary between exons 1 and 2 of transcript D (an RNA or RNA-derived cDNA, and / or an mRNA or mRNA-derived cDNA, preferably an RNA or RNA-derived cDNA).

[0097] The present invention provides a nucleic acid molecule as described above, which comprises or consists of a region that hybridizes to a segment present in transcripts D, G, and H (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably RNA or RNA), or a fragment or homolog thereof, that is not present in transcript A (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably RNA or RNA), or a fragment or homolog thereof, but further comprises a region (e.g., at least 15 contiguous nucleotides) that hybridizes to a segment present in transcript H (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably RNA or RNA), or a fragment or homolog thereof, that is not present in transcript D (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably RNA or RNA), or a fragment or homolog thereof. Such regions can be readily identified by comparing the sequences of the transcripts.For example, the segment can be 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 (i.e., at least about 90% identical) to a region (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) within exon 3' of transcript H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), a region spanning the boundary between exon 3 and exon 3' of transcript H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a region spanning the boundary between exon 3' and exon 4 of transcript H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA). It will be appreciated that such nucleic acid molecules will be designed to hybridize to a sufficient number of nucleotides in exon 3 and exon 3', respectively, or in exon 3' and exon 4, respectively, to distinguish them from other features in the HSD17B13 transcript (e.g., the boundary between exon 3 and exon 4). By way of example, the segment can comprise or consist of a region of transcript H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) within exon 3', spanning the boundary between exon 3 and exon 3', or spanning the boundary between exon 3' and exon 4.

[0098] The present invention provides a nucleic acid molecule as described above, which comprises or consists of a region that hybridizes to a segment present in transcripts D, G, and H (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably RNA or RNA), or a fragment or homolog thereof, that is not present in transcript A (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably RNA or RNA), or a fragment or homolog thereof, but further comprises a region (e.g., at least 15 contiguous nucleotides) that hybridizes to a segment present in transcript G (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably RNA or RNA), or a fragment or homolog thereof, that is not present in transcript D (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably RNA or RNA), or a fragment or homolog thereof. Such regions can be readily identified by comparing the sequences of the transcripts. For example, the segment can be 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 (i.e., at least about 90% identical) to a region spanning the boundary of exon 1 and exon 3 (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) of transcript G (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA). It is understood that such nucleic acid molecules will be designed to hybridize to a sufficient number of nucleotides in each of exon 1 and exon 3 to distinguish it from other features in the HSD17B13 transcript (e.g., the boundary between exon 1 and exon 2 or the boundary between exon 2 and exon 3).By way of example, the segment can include or consist of a region of transcript G (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA) spanning the boundary between exon 1 and exon 3.

[0099] Also provided is a nucleic acid molecule comprising or consisting of a region (e.g., at least 15 contiguous nucleotides) of a nucleic acid segment encoding the HSD17B13 protein that hybridizes to a segment present in transcript E (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, but not present in transcript A (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof. Such a region can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript E contains an additional exon between exon 3 and exon 4. Thus, the present invention provides a nucleic acid molecule that hybridizes to at least 15 consecutive nucleotides of a nucleic acid encoding an HSD17B13 protein, wherein the consecutive nucleotides are a region (e.g., at least 5 consecutive nucleotides, at least 10 consecutive nucleotides, or at least 15 consecutive nucleotides) within an exon 3' of transcript E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or RNA). A nucleic acid molecule comprising or consisting of a segment that is 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 (i.e., at least about 90% identical) to the region spanning the boundary between exon 3 and exon 3' of a cDNA derived from NA, preferably a cDNA derived from RNA or RNA, or the region spanning the boundary between exon 3' and exon 4 of transcript E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably a cDNA derived from RNA or RNA).It will be appreciated that such nucleic acid molecules will be designed to hybridize to a sufficient number of nucleotides in exon 3 and exon 3', respectively, or in exon 3' and exon 4, respectively, to distinguish them from other features in the HSD17B13 transcript (e.g., the boundary between exon 3 and exon 4). By way of example, the segment can comprise or consist of a region within exon 3' of transcript E (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), a region of transcript E (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) spanning the boundary between exon 3 and exon 3', or a region spanning the boundary between exon 3' and exon 4 (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA).

[0100] In some embodiments, nucleic acid molecules of the present disclosure further comprise or consist of a region (e.g., 15 contiguous nucleotides) that hybridizes to a segment present in transcript E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, that is not present in transcript H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof. Such segments can be readily identified by comparing the sequences of the transcripts. For example, a segment present in transcript E (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, that is not present in transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides), can be at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical (i.e., at least about 90% identical) to a region spanning the boundary between exon 6 and exon 7 of transcript E (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in order to distinguish it from transcript G. It is understood that such nucleic acid molecules would be designed to hybridize to a sufficient number of nucleotides in each of exon 6 and exon 7 to distinguish them from other features in the HSD17B13 transcript (particularly the additional guanine at the 3' end of exon 6 of transcript H).

[0101] Also provided is a nucleic acid molecule comprising or consisting of a region (e.g., at least 15 contiguous nucleotides) hybridizing to a segment of a nucleic acid encoding an HSD17B13 protein that is present in transcript F (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, but not present in transcript A (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof. Such a region can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript F contains a read-through region from exon 6 to intron 6. Thus, the present invention provides a nucleic acid molecule that hybridizes to at least 15 consecutive nucleotides of a nucleic acid encoding an HSD17B13 protein, wherein the consecutive nucleotides comprise or consist of a segment (e.g., at least 5 consecutive nucleotides, at least 10 consecutive nucleotides, or at least 15 consecutive nucleotides) that is 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 (i.e., at least about 90% identical) to a region within the read-through into intron 6 in transcript F (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) or to a region spanning the boundary between the read-through into intron 6 and the remainder of exon 6 in transcript F (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from RNA or RNA).It will be appreciated that such nucleic acid molecules will be designed to hybridize to a sufficient number of nucleotides in the readthrough region to distinguish it from other features in the HSD17B13 transcript (e.g., the boundary between exon 6 and exon 7 in other HSD17B13 transcripts). Optionally, the consecutive nucleotides comprise or consist of a sequence present in transcript F (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) that is not present in transcript F' (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) (i.e., a thymine insertion). Transcript F' also contains a readthrough region from exon 6 to intron 6 compared to transcript A, but the readthrough region does not contain the thymine insertion present in the HSD17B13 rs72613567 variant gene. By way of example, the segment may comprise or consist of a region of transcript F (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA) within the read-through into intron 6 or spanning the boundary between the read-through into intron 6 and the remainder of exon 6.

[0102] Also provided is a nucleic acid molecule comprising or consisting of a segment of a nucleic acid encoding an HSD17B13 protein, the segment hybridizing to a segment present in transcript F' (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, but not present in transcript A (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof (e.g., at least 15 contiguous nucleotides). Such a region can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript F' includes a read-through region from exon 6 to intron 6. Thus, provided are nucleic acid molecules that hybridize to at least 15 contiguous nucleotides of a nucleic acid encoding an HSD17B13 protein, wherein the contiguous nucleotides comprise or consist of a segment (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) that is 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 (i.e., at least about 90% identical) to a region within the read-through into intron 6 in transcript F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) or to a region spanning the boundary between the read-through into intron 6 and the remainder of exon 6 in transcript F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA).It will be appreciated that such nucleic acid molecules will be designed to hybridize to a sufficient number of nucleotides in the readthrough portion to distinguish it from other features in the HSD17B13 transcript (e.g., the boundary between exons 6 and 7 in other HSD17B13 transcripts). Optionally, the contiguous nucleotides comprise or consist of a sequence present in transcript F' (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) that is not present in transcript F (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA). The readthrough portion in transcript F contains a thymine insertion present in the HSD17B13 rs72613567 variant gene, whereas the readthrough portion in transcript F' does not contain this thymine insertion. By way of example, the segment may comprise or consist of a region of transcript F' (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA) within the read-through into intron 6 or spanning the boundary between the read-through into intron 6 and the remainder of exon 6.

[0103] Also provided are nucleic acid molecules comprising or consisting of a region (e.g., at least 15 contiguous nucleotides) of a nucleic acid segment encoding HSD17B13 protein that hybridizes to a segment present in transcript C (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof, but not present in transcript A (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a fragment or homolog thereof. Such a region can be readily identified by comparing the sequences of the transcripts. Compared to transcript A, transcript C lacks exon 6 (i.e., transcript C includes the boundary between exons 5 and 7, which is not present in transcript A). Thus, the present invention provides a nucleic acid molecule that hybridizes to at least 15 contiguous nucleotides of a nucleic acid encoding an HSD17B13 protein, wherein the contiguous nucleotides comprise or consist of a segment (e.g., at least 5 contiguous nucleotides, at least 10 contiguous nucleotides, or at least 15 contiguous nucleotides) that is 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 (i.e., at least about 90% identical) to the region spanning the boundary between exon 5 and exon 7 of transcript C (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA). It is understood that such nucleic acid molecules will be designed to hybridize to a sufficient number of nucleotides in exon 5 and exon 7 to distinguish them from other features in the HSD17B13 transcript (e.g., the boundaries between exons 5 and 6 or between exons 6 and 7 in other HSD17B13 transcripts).By way of example, the segment can include or consist of a region of transcript C (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA) spanning the boundary between exon 5 and exon 7.

[0104] The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are bound. 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 multiple types of probes are bound in an array, grid, or other organized pattern.

[0105] The solid-state substrate used in the solid support can include any solid material to which molecules can be bound. Examples of such materials include acrylamide, agarose, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, Teflon, fluorocarbon, nylon, silicone rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropyl fumerate, collagen, glycosaminoglycans, and polyamino acids. Solid-state substrates can be in any useful form, including thin films, membranes, bottles, dishes, fibers, woven fibers, shaped polymers, particles, beads, microparticles, or combinations. Solid-state substrates and solid supports can be porous or non-porous. The solid-state substrate can be in the form of a microtiter dish, such as a standard 96-well type. In some embodiments, multiwell glass slides, typically containing one array per well, can be used. In some embodiments, the support is a microarray.

[0106] The nucleic acid molecules disclosed herein can comprise RNA, DNA, or both RNA and DNA. The nucleic acid molecules disclosed herein can be linked or fused to a heterologous nucleotide sequence, such as in a vector, or linked or fused to a heterologous label. For example, the nucleic acid molecules disclosed herein can be in a vector or exogenous donor sequence containing the nucleic acid molecule and a heterologous nucleotide sequence. The nucleic acid molecules disclosed herein can also be linked or fused to a heterologous label, such as a fluorescent label. Other examples of labels are disclosed elsewhere herein.

[0107] 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. Examples of such labels include radioactive labels that can be measured with a radiation counter; pigments, dyes, or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (e.g., fluorophores) that generate an output signal upon excitation of a suitable molecular adduct and can be visualized by excitation with light absorbed by the dye or can be measured with a standard fluorometer or imaging system. Labels can also be, for example, chemiluminescent substances (where output signals are generated by chemical modification of a signal compound), metal-containing substances, or enzymes (where signal generation relies on the enzyme for secondary signal generation, such as the formation of a colored product from a colorless substrate). The term "label" can also refer to a "tag" or hapten that can be selectively attached to a conjugated molecule, such that the conjugated molecule generates a detectable signal upon addition of a substrate. For example, biotin can be used as a tag, followed by binding to the tag using an avidin or streptavidin conjugate of horseradish peroxidase (HRP), followed by detection of the presence of HRP using a calorimetric (e.g., tetramethylbenzidine (TMB)) or fluorogenic substrate. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG, 3XFLAG, 6XHis, polyhistidine, glutathione-S-transferase (GST), maltose-binding protein, epitope tags, or the Fc portion of an immunoglobulin. Many labels are known, including, for example, particles, fluorophores, haptens, enzymes and their calorimetric substrates, fluorogenic and chemiluminescent substrates, and other labels.

[0108] The nucleic acid molecules of the present disclosure can be modified nucleic acid molecules, and can include, for example, nucleotides, or non-natural or modified nucleotides (such as nucleotide analogs or nucleotide substitutes). Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or that have non-natural moieties introduced 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.

[0109] The nucleic acid molecules disclosed herein can also contain one or more nucleotide analogs or nucleotide substitutions. A nucleotide analog is a nucleotide containing a modification to either the base moiety, sugar moiety, 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 different purine or pyrimidine bases (e.g., pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl, etc.). 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 cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), Examples of suitable nucleotide analogs include, but are not limited to, uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including, but not limited to, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, and 5-methylcytosine), can enhance the stability of duplex formation. Base modifications, for example, in combination with sugar modifications (such as 2'-O-methoxyethyl), can often confer unique properties such as increased duplex stability.

[0110] Nucleotide analogs can also contain modifications to the sugar moiety, including, but not limited to, natural and synthetic modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, modifications at the 2' position of OH, F, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, or O-alkyl-O-alkyl, where these alkyls, alkenyls, and alkynyls are substituted or unsubstituted. 1-10 Alkyl or C 2-10 Alkenyl and C 2-10 Exemplary sugar modifications at 2' include -O[(CH) 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 CH3)]2 (wherein n and m are from 1 to about 10), but are not limited to these.

[0111] Other modifications at the 2' position include C 1-10Modified sugars include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides or groups for improving the pharmacological properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar in 3'-terminal nucleotides or 2'-5'-linked oligonucleotides and the 5' position of 5'-terminal nucleotides. Modified sugars can also include sugars containing modifications such as CH2 and S at the bridging ring oxygen. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar.

[0112] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, moieties that can modify the linkage between two nucleotides, including phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidate and aminoalkylphosphoramidate), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate or modified phosphate linkages between two nucleotides can be via a 3'-5' or 2'-5' linkage, and the linkage can include polarity inversions such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0113] Nucleotide substitutes also include nucleotides or nucleotide analogs in which the phosphate or sugar moiety has been replaced. In some embodiments, the nucleotide substitute may not contain a standard phosphorus atom. The phosphate substitution can be, for example, a short-chain alkyl or cycloalkyl internucleoside linkage, a mixed heteroatom and alkyl or cycloalkyl internucleoside linkage, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These substitutions include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other moieties containing a mixture of N, O, S, and CH2 moieties.

[0114] It is also understood that in nucleotide substitutes, both the sugar and phosphate moieties of the nucleotide can be replaced, for example, by an amide-type bond (aminoethylglycine) (PNA).

[0115] Other types of molecules (conjugates) can also be attached to nucleotides or nucleotide analogs, for example, to enhance cellular uptake.Conjugates can be chemically linked to nucleotides or nucleotide analogs.Such conjugates include, for example, lipid moieties (such as cholesterol moieties), cholic acid, thioethers (such as hexyl-S-tritylthiol), thiocholesterol, aliphatic chains (such as dodecanediol or undecyl residues), phospholipids (such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), polyamines or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0116] Also disclosed herein are polypeptides encoded by the nucleic acid molecules disclosed herein, and compositions comprising a nucleic acid or polypeptide disclosed herein and a carrier that enhances the stability of the isolated nucleic acid or protein (e.g., extends the period during which degradation products remain below a threshold level (e.g., less than 0.5% by weight of the initial nucleic acid or protein) under defined storage conditions (e.g., −20° C., 4° C., or ambient temperature), or enhances in vivo stability). Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cocrystals, and lipid microtubules.

[0117] The present invention also provides functional polynucleotides that can interact with the disclosed nucleic acid molecules. Functional polynucleotides are nucleic acid molecules that have a specific function, such as binding to a target molecule or catalyzing a specific reaction. 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, or stimulators of the specific activity of a target molecule, or functional polynucleotides can have de novo activity independent of any other molecule.

[0118] Antisense molecules are designed to interact with target nucleic acid molecules through either canonical or non-canonical base pairing. The interaction between the antisense molecule and the target molecule is designed to promote the destruction of the target molecule, for example, through RNase H-mediated degradation of RNA-DNA hybrids. Alternatively, antisense molecules are designed to interfere with the processing functions normally performed in the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are many methods for optimizing antisense efficiency by identifying the most accessible regions of the target molecule. Exemplary methods include, but are not limited to, in vitro selection experiments using DMS and DEPC and DNA modification tests. Antisense molecules generally exhibit a potent potency of approximately 10 -6 Below, about 10 -8 Below, about 10 -10 less than or about 10 -12 The following dissociation constant (k d ) that bind to a target molecule. Examples of antisense molecules include, but are not limited to, antisense RNA, small interfering RNA (siRNA), and short hairpin RNA (shRNA).

[0119] In some embodiments, any of the nucleic acid molecules, genomic DNA molecules, minigenes, RNA molecules, mRNA molecules, or cDNA molecules disclosed herein can be purified, e.g., at least about 90% pure. In some embodiments, any of the nucleic acid molecules disclosed herein can be purified, e.g., at least about 95% pure. In some embodiments, any of the nucleic acid molecules disclosed herein can be purified, e.g., at least about 99% pure. Purification is by human hands, using human-made purification techniques.

[0120] The present disclosure also provides vectors comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vectors of the present disclosure comprise any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vectors of the present disclosure can be viral or non-viral vectors capable of transporting nucleic acid molecules. In some embodiments, the vectors of the present disclosure are plasmids or cosmids. In some embodiments, the vectors of the present disclosure are viral vectors, where additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors of the present disclosure are capable of autonomous replication in a host cell into which they are introduced. In some embodiments, the vectors of the present disclosure can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain vectors can induce expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or "expression vectors." Such vectors can also be targeting vectors.

[0121] In some embodiments, proteins encoded by the various genetic variants disclosed herein are expressed by inserting a nucleic acid molecule encoding the disclosed genetic variant into an expression vector such that the gene is operably linked to expression control sequences, such as transcriptional and translational regulatory sequences. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art. In some embodiments, nucleic acid molecules comprising the disclosed genetic variants can be ligated into a vector such that the transcriptional and translational regulatory sequences within the vector perform their intended function of regulating the transcription and translation of the genetic variant.

[0122] In addition to a nucleotide sequence containing a disclosed genetic variant, the recombinant expression vector of the present disclosure can have a regulatory sequence that controls the expression of the genetic variant in a host cell, such as a promoter and / or enhancer derived from a retroviral LTR, a promoter and / or enhancer derived from cytomegalovirus (CMV) (e.g., the CMV promoter / enhancer), a promoter and / or enhancer derived from simian virus 40 (SV40) (e.g., the SV40 promoter / enhancer), a promoter and / or enhancer derived from adenovirus (e.g., the adenovirus major late promoter (AdMLP)), a polyoma promoter, and a strong mammalian promoter (e.g., native immunoglobulin and actin promoters). Methods for expressing polypeptides in bacterial or fungal cells (e.g., yeast cells) are also well known.

[0123] The promoter can 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).

[0124] In addition to nucleotide sequences containing the disclosed genetic variants and regulatory sequences, the recombinant expression vectors of the present disclosure can contain additional sequences, such as sequences that regulate replication of the vector in host cells, and selectable marker genes. Exemplary selectable marker genes include, but are not limited to, the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells with methotrexate selection / amplification), the Neo gene (for G418 selection), and the glutamate synthase (GS) gene.

[0125] The present disclosure also provides cells (e.g., recombinant host cells) containing any one or more of the nucleic acid molecules of the present disclosure and / or any one or more of the polypeptides disclosed herein, including vectors containing the nucleic acid molecules of the present disclosure. The cells can be in vitro, ex vivo, or in vivo. The nucleic acid molecules can be linked to promoters and other regulatory sequences such that they are expressed to produce the encoded proteins. Additionally, cell lines of such cells are provided.

[0126] In some embodiments, the cells of the present disclosure are totipotent or pluripotent cells (e.g., embryonic stem (ES) cells, such as rodent ES cells, mouse ES cells, or rat ES cells). Pluripotent and / or totipotent cells can be, for example, ES cells or ES-like cells, such as induced pluripotent stem (iPS) cells. According to the present disclosure, the embryonic stem cells can be non-human embryonic stem cells. In some embodiments, the cells of the present disclosure are primary somatic cells or non-primary somatic cells. Such cells can be isolated by conventional techniques and include, for example, somatic cells, hematopoietic cells, endothelial cells, epithelial cells, fibroblasts, mesenchymal cells, keratinocytes, melanocytes, monocytes, mononuclear cells, adipocytes, preadipocytes, neurons, glial cells, hepatocytes, skeletal myoblasts, and smooth muscle cells. For example, the primary cells can be derived from connective tissue, muscle tissue, nervous system tissue, or epithelial tissue.

[0127] In some embodiments, the cells of the present disclosure may not normally proliferate indefinitely, but may be mutated or modified to avoid normal cellular senescence and instead undergo continuous division. Such mutations or modifications may be naturally occurring or intentionally induced. Examples of immortalized cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney cells (e.g., HEK293 cells), and mouse embryonic fibroblast cells (e.g., 3T3 cells). In some embodiments, the cells of the present disclosure are differentiated cells, such as hepatocytes (e.g., human hepatocytes).

[0128] The cells of the present disclosure can be from any source. For example, the cells of the present disclosure can be eukaryotic cells, animal cells, plant cells, or fungal (e.g., yeast) cells. Such cells can be fish cells or avian cells, or such cells can be mammalian cells (such as human cells, non-human mammalian cells, rodent cells, mouse cells, or rat cells). Mammals include, but are not limited to, humans, non-human primates, monkeys, apes, cats, dogs, horses, steers, deer, bison, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs), livestock (e.g., bovine species (e.g., cows, steers), ovine (e.g., sheep, goats), and porcine (e.g., pigs, wild boars). Avian includes, but is not limited to, chickens, turkeys, ostriches, geese, ducks, and the like. Domestic and farm animals are also included. The term "non-human animal" does not include humans. In some embodiments, the cells of the present disclosure are human cells.

[0129] The present disclosure provides for the use of any of the nucleic acid molecules described herein as probes or primers for detecting a variant HSD17B13 gene or variant HSD17B13 transcript, for determining a human subject's susceptibility or risk for developing liver disease, or for determining a human subject's risk for progressing to a clinically more advanced stage of fatty liver disease.

[0130] The present disclosure provides HSD17B13 isoform polypeptides and fragments thereof, particularly HSD17B13 isoform polypeptides and fragments thereof produced by the HSD17B13 rs72613567 variant.

[0131] The present disclosure provides polypeptides comprising or consisting of an amino acid sequence 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 the amino acid sequence of HSD17B13 isoform D (SEQ ID NO:42). In some embodiments, the polypeptide comprises or consists of an amino acid sequence at least about 90% identical to the amino acid sequence of HSD17B13 isoform D (SEQ ID NO:42). In some embodiments, the polypeptides of the present disclosure comprise or consist of the amino acid sequence of SEQ ID NO:42. In some embodiments, these polypeptides have 274 amino acids. In some embodiments, these polypeptides have Val-Ser-Ser at the C-terminus. In some embodiments, these polypeptides are associated with a reduced risk of developing any of the liver diseases described herein or a reduced risk of progressing to a more clinically advanced stage of fatty liver disease.

[0132] In some embodiments, a polypeptide of the disclosure comprises or consists of an amino acid sequence that is 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 (i.e., at least about 90% identical) to isoform A, isoform B, isoform C, isoform D, isoform E, isoform F, isoform F', isoform G, or isoform H. In some embodiments, the HSD17B13 protein is isoform A, isoform B, isoform C, isoform D, isoform E, isoform F, isoform F', isoform G, or isoform H.

[0133] In some embodiments, a polypeptide of the disclosure comprises or consists of an amino acid sequence that is 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 (i.e., at least about 90% identical) to isoform C, isoform D, isoform E, isoform F, isoform F', isoform G, or isoform H. In some embodiments, a polypeptide of the disclosure is isoform C, isoform D, isoform E, isoform F, isoform F', isoform G, or isoform H.

[0134] In some embodiments, a polypeptide of the disclosure comprises or consists of an amino acid sequence that is 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 (or at least about 90%) to isoform C, isoform D, isoform F, isoform G, or isoform H. In some embodiments, a polypeptide of the disclosure is isoform C, isoform D, isoform F, isoform G, or isoform H.

[0135] In some embodiments, a polypeptide of the disclosure comprises or consists of an amino acid sequence that is 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 isoform D. In some embodiments, a polypeptide of the disclosure comprises or consists of an amino acid sequence that is at least about 90% identical to isoform D. In some embodiments, a polypeptide of the disclosure is isoform D.

[0136] In some embodiments, a polypeptide of the disclosure comprises or consists of, for example, at least 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 consecutive amino acids of: i) HSD17B13 isoform A, B, C, D, E, F, F', G, or H, or a fragment thereof; ii) HSD17B13 isoform C, D, E, F, F', G, or H, or a fragment thereof; iii) HSD17B13 isoform C, D, F, G, or H, or a fragment thereof; or iv) HSD17B13 isoform D, or a fragment thereof. It is understood that gene sequences within a population, and the proteins encoded by such genes, can vary due to polymorphisms, such as single nucleotide polymorphisms. The sequences shown herein for each HSD17B13 isoform are merely exemplary sequences. Other sequences are possible.

[0137] As an example, a polypeptide of the present disclosure can comprise or consist of a segment (e.g., at least 8 contiguous amino acids) that is 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 (i.e., at least about 90% identical) to a segment comprising at least a portion of a region encoded by exon 7 in isoform D, isoform G, or isoform H (or a fragment or homolog thereof) that is not present in isoform A (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the isoforms. The region encoded by exon 7 in isoforms D, G, and H is frameshifted and truncated compared to the region encoded by exon 7 in isoform A.

[0138] Such polypeptides can further comprise or consist of a segment present in isoform D (or a fragment or homolog thereof) that is absent in isoform G (or a fragment or homolog thereof), and can further comprise a segment present in isoform D (or a fragment or homolog thereof) that is absent in isoform H (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the isoforms. For example, such polypeptides can comprise or consist of a segment of contiguous amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical (i.e., at least about 90% identical) to a segment of isoform D spanning the boundary of the region encoded by exon 3 and exon 4, in order to distinguish it from isoform H. Similarly, such polypeptides can comprise or consist of a segment of contiguous amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical (i.e., at least about 90% identical) to a segment within the region encoded by exon 2 in isoform D, a segment spanning the boundary between the region encoded by exon 1 and exon 2 in isoform D, or a segment spanning the boundary between the region encoded by exon 2 and exon 3 in isoform D, in order to distinguish it from isoform G.

[0139] Like isoform D, the region encoded by exon 7 in isoform H is frameshifted and truncated compared to isoform A. However, in addition, isoform H contains a region encoded by an additional exon (exon 3') between exon 3 and exon 4 compared to isoforms A and D. Thus, such polypeptides can include or consist of segments present in isoforms D, G, and H (or fragments or homologs thereof) that are absent in isoform A (or fragments or homologs), as described above, but can also include a segment (e.g., at least 8 contiguous amino acids) from isoform H (or fragments or homologs thereof) that is absent in isoform D (or fragments or homologs thereof). Such regions can be readily identified by comparing the sequences of the isoforms. For example, such a polypeptide can further comprise or consist of a segment of contiguous amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) that is 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 (i.e., at least about 90% identical) to a segment comprising at least a portion of the region encoded by exon 3' in isoform H.

[0140] Like isoform D, the region encoded by exon 7 in isoform G is frameshifted and truncated compared to isoform A. However, in addition, isoform G contains the boundary between exon 1 and exon 3, which is absent in isoforms A and D, due to the deletion of the region encoded by exon 2 compared to isoforms A and D. Thus, such polypeptides can include or consist of segments present in isoforms D, G, and H (or fragments or homologs thereof) that are absent in isoform A (or fragments or homologs), as described above, but can also include a segment (e.g., at least 8 contiguous amino acids) from isoform G (or fragments or homologs thereof) that is absent in isoform D (or fragments or homologs thereof). Such regions can be readily identified by comparing the sequences of the isoforms. For example, such polypeptides can further comprise or consist of a segment of contiguous amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) that is 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 (i.e., at least about 90% identical) to a segment spanning the boundary of the region encoded by exon 1 and exon 3 in isoform G.

[0141] Also provided herein are polypeptides comprising or consisting of a segment (e.g., at least 8 contiguous amino acids) present in isoform E (or a fragment or homolog thereof) that is not present in isoform A (or a fragment or homolog thereof). Isoform E contains a region encoded by an additional exon (exon 3') between exon 3 and exon 4 that is not present in isoform A. Such regions can be readily identified by comparing the sequences of the isoforms. Thus, a polypeptide of the present disclosure can comprise or consist of at least 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 consecutive amino acids of an HSD17B13 isoform protein (e.g., at least 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 15 consecutive amino acids of an HSD17B13 protein), and the consecutive amino acids can be A segment of amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) is 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 (i.e., at least about 90% identical) to a segment comprising at least a portion of the region encoded by exon 3' in isoform E or isoform H. Optionally, such a polypeptide can further comprise or alternatively consist of a segment (e.g., at least 8 contiguous amino acids) from isoform E (or a fragment or homolog thereof) that is not present in isoform H (or a fragment or homolog thereof). Such regions can be readily identified by comparing the sequences of the isoforms.For example, such polypeptides can further comprise or alternatively consist of a segment of contiguous amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) that is 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 (i.e., at least about 90% identical) to a segment spanning the boundary of the region encoded by exon 6 and exon 7 of isoform E.

[0142] Also provided herein is a polypeptide comprising or consisting of a segment (e.g., at least 8 contiguous amino acids) present in isoform F (or a fragment or homolog thereof) that is not present in isoform A (or a fragment or homolog thereof). Isoform F contains a region encoded by the read-through portion from exon 6 to intron 6 that is not present in isoform A. Such a region can be readily identified by comparing the sequences of the isoforms. Thus, a polypeptide of the disclosure can comprise or consist of at least 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 consecutive amino acids of an HSD17B13 isoform protein (e.g., at least 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 15 consecutive amino acids of an HSD17B13 protein), and the consecutive amino acids can be A segment of amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) is 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 (i.e., at least about 90% identical) to a segment of isoform F that includes at least a portion of the region encoded by the read-through into intron 6.

[0143] The present invention provides a polypeptide comprising or consisting of a segment (e.g., at least 8 contiguous amino acids) present in isoform C (or a fragment or homolog thereof) that is not present in isoform A (or a fragment or homolog thereof). Compared to isoform A, isoform C lacks the region encoded by exon 6 and includes the boundary between exons 5 and 7, which is not present in isoform A. Such regions can be readily identified by comparing the sequences of the isoforms. Thus, a polypeptide of the disclosure can comprise at least 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 22, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 consecutive amino acids of an HSD17B13 protein isoform (e.g., at least 8 consecutive amino acids, at least 10 consecutive amino acids, or at least 15 consecutive amino acids of an HSD17B13 protein), and the consecutive amino acids can be fused together. A segment of amino acids (e.g., at least 3 contiguous amino acids, at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least 10 contiguous amino acids, or at least 15 contiguous amino acids) is 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 (i.e., at least about 90% identical) to a segment spanning the boundary of the region encoded by exon 5 and exon 7 in isoform C.

[0144] Any of the isolated polypeptides disclosed herein can be linked to a heterologous molecule or heterologous tag. Examples of such heterologous molecules or tags are disclosed elsewhere herein. For example, the heterologous molecule can be an immunoglobulin Fc domain, a peptide tag as disclosed elsewhere herein, poly(ethylene glycol), polysialic acid, or glycolic acid.

[0145] The present disclosure also provides methods for producing any of the polypeptides or fragments thereof disclosed herein. For example, a polypeptide or fragment thereof can be produced from a host cell containing a nucleic acid molecule (e.g., a recombinant expression vector) encoding the polypeptide or fragment thereof. Such methods can include producing the polypeptide or fragment thereof by culturing a host cell containing a nucleic acid molecule (e.g., a recombinant expression vector) encoding the polypeptide or fragment thereof under conditions sufficient to produce the polypeptide or fragment thereof. The nucleic acid can be operably linked to a promoter active in the host cell, and culturing can be under conditions to express the nucleic acid. Such methods can further include recovering the expressed polypeptide or fragment thereof. The recovery can further include purifying the polypeptide or fragment thereof.

[0146] Examples of suitable protein expression systems include host cells such as bacterial cell expression systems (e.g., Escherichia coli, Lactococcus lactis), yeast cell expression systems (e.g., Saccharomyces cerevisiae, Pichia pastoris), insect cell expression systems (e.g., baculovirus-mediated protein expression), and mammalian cell expression systems.

[0147] In some embodiments, the nucleic acid molecule encodes a tag in-frame with the polypeptide or fragment thereof to facilitate purification of the protein. Examples of tags are disclosed elsewhere herein. Such a tag can, for example, bind to a partner ligand (e.g., a partner ligand immobilized on a resin) to allow the tagged protein to be isolated from all other proteins (e.g., host cell proteins).

[0148] Other methods can also be used to produce polypeptides or fragments thereof. For example, two or more peptides or polypeptides can be linked together using protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) chemistries. Alternatively, peptides or polypeptides can be independently synthesized in vivo as described herein. Once isolated, these independent peptides or polypeptides can be linked to form peptides or fragments thereof via similar peptide condensation reactions.

[0149] In some embodiments, polypeptides of the present disclosure can have post-expression modifications such as, for example, glycosylation, acetylation, and phosphorylation, as well as other modifications (both naturally occurring and non-naturally occurring) known in the art. A polypeptide can be an entire protein or a subsequence thereof.

[0150] The present disclosure also provides a method of making any of the polypeptides disclosed herein, comprising producing the polypeptide by culturing a host cell containing a recombinant expression vector comprising a nucleic acid molecule that comprises a polynucleotide capable of encoding one or more of the polypeptides disclosed herein or their complements.

[0151] The polypeptides disclosed herein can include the amino acid sequence of a naturally occurring HSD17B13 isoform protein, or can include a non-naturally occurring sequence. In one example, the non-naturally occurring sequence can differ from the non-naturally occurring sequence by conservative amino acid substitutions. For example, the sequence can be identical except for the conservative amino acid substitutions.

[0152] Any of the polypeptides disclosed herein can further have one or more substitutions (e.g., conservative amino acid substitutions), insertions, or deletions. Insertions include, for example, amino- or carboxyl-terminal fusions and intrasequence insertions of one or more amino acid residues. Techniques for making substitutions at predetermined sites in DNA with a known sequence are well known (e.g., M13 primer mutagenesis and PCR mutagenesis). Amino acid substitutions are typically of a single residue, but can be made at many different positions at once; insertions are usually on the scale of about 1-10 amino acid residues, and deletions range from about 1-30 residues. Deletions or insertions can be made in adjacent pairs (i.e., deletion of two residues or insertion of two residues). Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final construct. In some embodiments, these mutations do not place the sequence out of reading frame and do not create complementary regions that could result in secondary mRNA structure.

[0153] In some embodiments, the polypeptides disclosed herein are linked or fused to heterologous polypeptides, or heterologous molecules or labels, examples of which are numerously disclosed elsewhere herein. For example, proteins of the present disclosure can be fused to heterologous polypeptides that improve or decrease stability. The fused domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or internally of the polypeptide. The fusion partner may, for example, assist in providing a T-helper epitope (immunological fusion partner) or assist in expressing the polypeptide at a higher yield than the native recombinant polypeptide (expression enhancer). Certain fusion partners are both immunological and expression-enhancing fusion partners. Other fusion partners may be selected to improve the solubility of the polypeptide or to facilitate targeting of the polypeptide to a desired intracellular compartment. Some fusion partners contain affinity tags to facilitate purification of the polypeptide.

[0154] In some embodiments, the fusion protein is directly fused to the heterologous molecule or is linked to the heterologous molecule via a linker, such as a peptide linker. For example, a peptide linker sequence may include Gly, Asn, and Ser residues. Other near-neutral amino acids (such as Thr and Ala) may also be used in the linker sequence. Linker sequences may generally be, for example, from 1 to about 50 amino acids in length. A linker sequence is generally not necessary when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains or prevent steric interference.

[0155] In some embodiments, the polypeptide of the present disclosure is operably linked to a cell penetration domain.For example, the cell penetration domain can be derived from HIV-1 TAT protein, TLM cell penetration motif from human hepatitis B virus, MPG, Pep-1, VP22, cell penetration peptide from herpes simplex virus, or polyarginine peptide sequence.The cell penetration domain can be located at the N-terminus, C-terminus, or any position of the protein.

[0156] In some embodiments, the polypeptides of the present disclosure can be operably linked to a heterologous polypeptide, such as a fluorescent protein, purification tag, or epitope tag, to facilitate tracking or purification. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed), and the like. Monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein. Examples of tags include, but are not limited to, glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag1, Softag3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin.In some embodiments, the heterologous molecule is an immunoglobulin Fc domain, a peptide tag, a transduction domain, poly(ethylene glycol), polysialic acid, or glycolic acid.

[0157] In some embodiments, the isolated polypeptides contain non-naturally occurring or modified amino acids or peptide analogs. For example, D-amino acids or amino acids with functional substituents different from the naturally occurring amino acids are numerous. Reverse stereoisomers of natural peptides and stereoisomers of peptide analogs are disclosed.

[0158] In some embodiments, the isolated polypeptides of the present disclosure are peptidomimetics, which can be made to resemble peptides but are not linked via natural peptide bonds. For example, amino acid or amino acid analog linkages include, but are not limited to, -CHNH-, -CHS-, -CH-, -CH=CH- (cis and trans), -COCH-, -CH(OH)CH-, and -CHHSO-. Peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, etc.

[0159] In some embodiments, polypeptides of the present disclosure contain D-amino acids, which can be used to generate more stable peptides because D-amino acids are not recognized by peptidases. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., replacing L-lysine with D-lysine) can be used to generate more stable peptides. Cysteine ​​residues can be used to cyclize or link two or more peptides together.

[0160] The present disclosure also provides nucleic acid molecules encoding any of the polypeptides disclosed herein, including all degenerate sequences related to a particular polypeptide sequence (all nucleic acid molecules having a sequence that encodes a particular polypeptide sequence, and all nucleic acids (including degenerate nucleic acids) that encode the disclosed variants and derivatives of that protein sequence). Thus, while every specific nucleic acid sequence cannot be written out herein, in practice, every sequence is disclosed and described herein through the disclosed polypeptide sequences.

[0161] Percent identity (or percent complementarity) between specific regions of a nucleotide sequence within a nucleic acid molecule or an amino acid sequence within a polypeptide can be routinely determined using the BLAST program (basic alignment search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison, Wis.) with default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When percent sequence identity is referred to herein, higher percent sequence identity is preferred over lower percent sequence identity.

[0162] The present disclosure also provides compositions comprising any one or more of the nucleic acid molecules disclosed herein and / or any one or more of the polypeptides disclosed herein and a carrier and / or excipient. In some embodiments, the carrier improves the stability of the nucleic acid molecules and / or polypeptides disclosed herein (e.g., by extending the period during which degradation products remain below a threshold value (e.g., less than 0.5% by weight of the initial nucleic acid or protein) under certain storage conditions (e.g., −20° C., 4° C., or ambient temperature) or by improving in vivo stability). Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, etc.

[0163] The nucleic acid molecules and polypeptides disclosed herein can be introduced into cells by any means. Non-limiting transfection methods include chemical-based transfection methods using liposomes, nanoparticles, calcium, dendrimers, and cationic polymers (such as DEAE-dextran or polyethyleneimine). Viral methods can also be used for transfection (e.g., adenovirus, adeno-associated virus, lentivirus, retrovirus, transfection, lipid-mediated transfection, or nucleofection). In some embodiments, nucleofection is performed using the LONZA® NUCLEOFECTOR™ system. Introduction of nucleic acid molecules or proteins into cells can also be performed by microinjection. Non-chemical methods include electroporation, sonoporation, optical transfection, particle-based transfection (including gene gun or magnetic transfection), and intracellular injection. Introduction of nucleic acid molecules and proteins into cells can also be performed by hydrodynamic delivery (HDD). In some embodiments, nucleic acids or proteins can be introduced into cells in a carrier such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cocrystals, or lipid microtubules.

[0164] The nucleic acid molecule or protein can be introduced into the cell once or multiple times over a period of time, for example at least 2 times over a period of time, at least 3 times over a period of time, at least 4 times over a period of time, at least 5 times over a period of time, at least 6 times over a period of time, at least 7 times over a period of time, at least 8 times over a period of time, at least 9 times over a period of time, at least 10 times over a period of time, at least 11 times, at least 12 times over a period of time, at least 13 times over a period of time, at least 14 times over a period of time, at least 15 times over a period of time, at least 16 times over a period of time, at least 17 times over a period of time, at least 18 times over a period of time, at least 19 times over a period of time, or at least 20 times over a period of time.

[0165] The present disclosure provides methods for detecting the presence of a variant HSD17B13 rs72613567 gene in a biological sample containing genomic DNA, for detecting the presence or level of any one or combination of HSD17B13 transcripts C, D, E, F, F', G, and H, particularly D, in a biological sample containing RNA or cDNA derived from RNA, or mRNA or cDNA derived from mRNA, or for detecting the presence or level of any one or combination of HSD17B13 protein isoforms C, D, E, F, F', G, or H, particularly D, in a biological sample containing protein. It is recognized that gene sequences within a population, and the RNA, mRNA, and proteins encoded by those genes, can vary due to polymorphisms, such as single nucleotide polymorphisms. The sequences shown herein for the HSD17B13 gene, each HSD17B13 transcript, and each HSD17B13 isoform are merely exemplary sequences for the HSD17B13 gene, each HSD17B13 transcript (RNA, mRNA, and cDNA derived therefrom), and each HSD17B13 isoform. Other sequences for the HSD17B13 gene, each HSD17B13 transcript, and each HSD17B13 isoform are also possible.

[0166] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The sample may include any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy specimen, a fine needle aspiration biopsy specimen, or a sample of bodily fluid (such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine). In some cases, the sample includes a buccal swab. The sample used in the methods disclosed herein will vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample.

[0167] Biological samples can be subjected to different treatments depending on the assay used.For example, when detecting HSD17B13 rs72613567 variant nucleic acid molecules, preliminary treatments designed to isolate or enrich the genomic DNA of the sample can be used.For this purpose, various known techniques can be used.When detecting the mRNA level of HSD17B13 transcript C, D, E, F, G or H, various techniques can be used to enrich the mRNA of biological samples.Various methods can be used to detect the presence or level of specific HSD17B13 rs72613567 variant nucleic acid molecules.

[0168] The present disclosure provides methods for detecting a variant HSD17B13 rs72613567 gene in a cell or a subject (such as a human subject). The disclosure provides methods for detecting a variant HSD17B13 gene in a human subject, the method comprising or consisting of performing an assay on a biological sample from the human subject to determine whether a wild-type HSD17B13 gene has a thymine inserted between positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1, or whether a variant HSD17B13 gene has a thymine at position corresponding to position 12666 of SEQ ID NO: 2, wherein the presence of the thymine indicates a variant HSD17B13 gene. In some embodiments, the assay comprises or consists of sequencing a portion of the HSD17B13 gene including positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1, or including position 12666 of SEQ ID NO: 2. In some embodiments, the assay comprises, or consists of: i) contacting the biological sample with a primer that hybridizes to a region of the HSD17B13 gene that is within 50 nucleotides of positions 12665 ​​and 12666 of SEQ ID NO:1 or within 50 nucleotides of positions 12666 of SEQ ID NO:2 in the HSD17B13 gene; ii) extending the primer to at least pass through positions 12665 ​​and 12666 of SEQ ID NO:1 or positions 12666 of SEQ ID NO:2 in the HSD17B13 gene; and iii) determining whether the extension product of the primer contains a thymine inserted between positions 12665 ​​and 12666 of SEQ ID NO:1 in the wild-type HSD17B13 gene or a thymine present at position 12666 of SEQ ID NO:2 in the variant HSD17B13 gene. In some embodiments, the method further comprises determining whether the human subject is homozygous for a variant HSD17B13 gene.

[0169] In some embodiments, the methods of the present disclosure comprise, or consist of, for example, collecting a biological sample containing the HSD17B13 gene from a subject, and performing an assay on the biological sample to determine whether the position corresponding to position 12666 of SEQ ID NO: 2 in a variant HSD17B13 gene is occupied by thymine, or whether a thymine has been inserted between positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1 in a wild-type HSD17B13 gene. Determining that the position in the HSD17B13 gene corresponding to position 12666 of SEQ ID NO: 2 is occupied by thymine refers to determining the number of types of nucleotides sufficient at positions between positions 12665 ​​and 12666 of SEQ ID NO: 1 to determine whether a thymine has been inserted between positions 12665 ​​and 12666 of SEQ ID NO: 1. Such assays can include, for example, determining the presence of a nucleotide sequence at a position corresponding to position 12666 of SEQ ID NO:2 of the variant HSD17B13 gene (or positions 12665 ​​and 12666 of SEQ ID NO:1 of the wild-type HSD17B13 gene) and one or more surrounding positions (e.g., at least one, two, three, four, five, six, seven, eight, nine, or ten positions adjacent to one or each side of position 12666 of SEQ ID NO:2 of the variant HSD17B13 gene or positions 12665 ​​and 12666 of SEQ ID NO:1 of the wild-type HSD17B13 gene).

[0170] Assaying in such a method can include, for example, sequencing a portion of the HSD17B13 gene, including a position corresponding to position 12666 or positions 12666 and 12667 of SEQ ID NO: 2. Similarly, the assay can include sequencing a portion of the HSD17B13 gene, including a position corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1. For example, this method may include: i) contacting a biological sample with a primer (variation-specific primer) that hybridizes to a segment of the HSD17B13 gene that is adjacent to a position in the HSD17B13 gene corresponding to position 12666 or positions 12666 and 12667 of SEQ ID NO:2; ii) extending the primer so that it passes through at least a position in the HSD17B13 gene that corresponds to position 12666 or positions 12666 and 12667 of SEQ ID NO:2; and iii) determining which of the extension products of the primer is at a position in the HSD17B13 gene that corresponds to position 12666 or positions 12666 and 12667 of SEQ ID NO:2. As another example, this method may include: i) contacting a biological sample with a primer (e.g., a variation-specific primer) that hybridizes to a segment of the HSD17B13 gene that is adjacent to positions 12665 ​​and 12666 of SEQ ID NO: 1 in the HSD17B13 gene; ii) extending the primer so that it passes through at least positions 12665 ​​and 12666 of SEQ ID NO: 2 in the HSD17B13 gene; and iii) determining whether a thymine is present in the extension product of the primer between positions 12665 ​​and 12666 of the HSD17B13 gene that correspond to positions 12665 ​​and 12666 of SEQ ID NO: 1. In some embodiments, the variation-specific probe or variation-specific primer comprises or consists of a nucleotide sequence that is complementary to and / or hybridizes or specifically hybridizes to a particular HSD17B13 gene or transcript (such as transcript D), but does not hybridize or specifically hybridize to the wild-type HSD17B13 gene (SEQ ID NO: 1).As used herein, "close to" means within about 50 nucleotides, within about 45 nucleotides, within about 40 nucleotides, within about 35 nucleotides, within about 30 nucleotides, within about 25 nucleotides, within about 20 nucleotides, within about 15 nucleotides, within about 10 nucleotides, or within about 5 nucleotides of the particular position indicated.

[0171] Alternatively, the assay in such a method can include contacting the biological sample with a primer or probe that specifically hybridizes (e.g., under stringent conditions) to the HSD17B13 rs72613567 variant but does not specifically hybridize to the corresponding wild-type HSD17B13 sequence, and determining whether hybridization has occurred.

[0172] The present disclosure provides methods for detecting the presence of an HSD17B13 transcript in a human subject. The present disclosure provides methods for detecting the presence of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) in a human subject, comprising or consisting of performing an assay on a biological sample obtained from the subject, whereby the presence of HSD17B13 transcript D in the biological sample is determined. In some embodiments, the assay comprises or consists of contacting the biological sample with one or more primers or probes that specifically hybridize to a nucleic acid sequence of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or its complement, and determining whether hybridization has occurred. In some embodiments, the method comprises: i) a nucleotide sequence that is 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 (i.e., at least about 90% identical) to the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or a complement thereof; ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA); and / or iii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D (RNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA). It further comprises specifically detecting transcription product D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) by using a nucleic acid molecule comprising or consisting of about 5 nucleotides up to about 50 nucleotides that comprises or consists of a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of product D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA).In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 (i.e., at least about 90% identical) to SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the one or more primers or probes specifically hybridize to SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, and / or SEQ ID NO: 33. In some embodiments, the assay comprises reverse transcription polymerase chain reaction (RT-PCR). In some embodiments, the assay comprises sequencing.

[0173] The present disclosure provides methods for detecting the presence of one or a combination of HSD17B13 transcripts C, D, E, F, F', G, or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) in a cell or a subject (such as a human subject). Such methods can comprise, or consist of, for example, obtaining a biological sample from the subject that contains RNA or cDNA derived from RNA, or that contains cDNA derived from mRNA or mRNA, and performing an assay on the sample to determine the presence of transcript C, D, E, F, F', G, or H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) in the subject. For example, such an assay can detect a region or combination of regions present in one or more of transcripts C, D, E, F, F', G, or H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) that is absent in transcripts A and B (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) (e.g., a region or combination of regions that is unique to one or more of transcripts C, D, E, F, F', G, or H). Such regions may be unique to a particular transcript (e.g., unique to transcript C (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA)) or unique to a combination of transcripts (e.g., unique to transcripts D, G, and H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA)). Such regions can be readily identified by comparing the sequences of transcripts A through H, and are described in further detail elsewhere herein.

[0174] As an example, the assay can include RNA sequencing (RNA-Seq). As another example, the assay can include or consist of contacting the biological sample with one or more primers or probes that specifically hybridize to one or more sequences (combinations of which are unique to one or a combination of HSD17B13 transcripts A, B, C, D, E, F, F', G, or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) among transcripts C, D, E, F, F', G, and H (i.e., not present in transcripts A and B (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA)) and determining whether hybridization has occurred. Optionally, the assay can include reverse transcription polymerase chain reaction (RT-PCR). Assays such as those described above can be , can be specific for a particular HSD17B13 transcript or a particular combination of HSD17B13 transcripts. For example, transcripts D, G, and H contain an additional guanine inserted at the 3' end of exon 6 compared to transcripts A, B, and E, respectively (transcript C does not contain exon 6), and transcripts D, G, and H each contain exon 7, while transcript F reads through from exon 6 to intron 6. Thus, a primer or probe that hybridizes to a region spanning the boundary between exon 6 and exon 7 of transcript D (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA) can specifically detect the combination of transcripts D, G, and H (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA).It will be appreciated that such primers or probes will be designed to hybridize to a sufficient number of nucleotides in each of exon 6 and exon 7 to distinguish between an inserted guanine and other features in the HSD17B13 transcript (e.g., a read-through into intron 6 in transcript F (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA) or a deleted exon 6 in transcript C (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA)). Similarly, transcripts E and H each include exon 3' relative to all other transcripts. Thus, a primer or probe that specifically hybridizes to a region within exon 3' or to the boundary between exon 3' and exon 3 or exon 4 can specifically detect the combination of transcripts E and H (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA). It will be appreciated that such primers or probes will be designed to hybridize to a sufficient number of nucleotides in exon 3 and exon 3', respectively, or in exon 3' and exon 4, respectively, to distinguish from other features in the HSD17B13 transcript (e.g., the boundary between exon 3 and exon 4). Similarly, transcripts B and G each lack exon 2. Thus, a primer or probe that specifically hybridizes to a region spanning the boundary between exon 1 and exon 3 can specifically detect the combination of transcripts B and G (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA).It is understood that primers or probes will be designed to hybridize to a sufficient number of nucleotides in each of exon 1 and exon 3 to distinguish them from other features in the HSD17B13 transcript (e.g., the boundary between exon 1 and exon 2 or the boundary between exon 2 and exon 3).

[0175] In one embodiment, the one or more primers or probes specifically hybridize to a region spanning the boundary between exon 6 and exon 7 in transcript D (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), transcript G (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or transcript H (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) (i.e., containing an additional guanine at the 3' end of exon 6 (not present in exon 6 of other HSD17B13 transcripts)). Optionally, the one or more primers or probes further specifically hybridize to a region within exon 3' of transcript H (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), a region spanning the boundary between exon 3 and exon 3' of transcript H (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or a region spanning the boundary between exon 3' and exon 4 of transcript H (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or further specifically hybridize to a region spanning the boundary between exon 1 and exon 3 of transcript G (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA). Optionally, the one or more primers or probes further specifically hybridize to a region within exon 1 of any of transcripts A to H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA) (a region common to transcripts A to H).For example, the intervening sequence can be amplified using a primer that specifically hybridizes to a region within exon 1 of any of transcripts A to H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) and a primer that specifically hybridizes to a region spanning the boundary between exons 6 and 7 of transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), transcript G (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), and transcript D, transcript G, and transcript H can be distinguished based on the size of the amplified product. This is because transcript G lacks exon 2 compared to transcript D, and transcript H contains an additional exon between exon 3 and exon 4 compared to transcript D.

[0176] In another embodiment, the one or more primers or probes are directed to a region within an exon 3' of transcript E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) or transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or to a region within an exon 3' of transcript E (RNA or cDNA derived from ... H (R It specifically hybridizes to the region spanning the boundary between exon 3 and exon 3' of transcript E (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or to the region spanning the boundary between exon 3' and exon 4 of transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA). Optionally, the one or more primers or probes can further specifically hybridize to a region spanning the boundary between exon 6 and exon 7 of transcript D (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), transcript G (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or transcript H (RNA or cDNA derived from RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA).Alternatively, one or more primers or probes may also specifically hybridize to a region spanning the boundary between exon 6 and exon 7 of transcript E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA).For example, a region within exon 3' of transcript E (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) or transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), a region spanning the boundary between exon 3 and exon 3' of transcript E (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) or transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), Transcript E can be distinguished from transcript H by amplifying the intervening sequence using a primer that specifically hybridizes to a region spanning the boundary between exon 3' and exon 4 of transcript D (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA), or transcript H (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA), and a primer that specifically hybridizes to a region spanning the boundary between exon 6 and exon 7 of transcript D (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA), transcript G (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA), or transcript H (RNA or RNA-derived cDNA and / or mRNA or mRNA-derived cDNA, preferably RNA or RNA-derived cDNA). Only transcript H contains an additional guanine at the 3' end of exon 6, whereas transcript E does not.

[0177] In another embodiment, the one or more primers or probes specifically hybridize to a region within the readthrough into intron 6 of transcript F (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or to a region spanning the boundary between the readthrough into intron 6 of transcript F (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) and the remainder of exon 6. It will be appreciated that such primers or probes will be designed to hybridize to a sufficient number of nucleotides in the readthrough portion to distinguish this readthrough portion from other features in the HSD17B13 transcript (e.g., the boundary between exon 6 and exon 7 in other HSD17B13 transcripts). Optionally, the consecutive nucleotides include a sequence (i.e., a thymine insertion) that is present in transcript F (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) but is not present in transcript F' (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA). Transcript F' also includes a read-through region from exon 6 to intron 6 compared to transcript A, but the read-through region does not include the thymine insertion that is present in the HSD17B13 rs72613567 variant gene.

[0178] In another embodiment, one or more primers or probes specifically hybridize to a region within the readthrough into intron 6 in transcript F' (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) or to a region spanning the boundary between the readthrough into intron 6 and the remainder of exon 6 in transcript F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA). It is understood that such primers or probes will be designed to hybridize to a sufficient number of nucleotides in the readthrough portion to distinguish this readthrough portion from other features in the HSD17B13 transcript (e.g., the boundary between exon 6 and exon 7 in other HSD17B13 transcripts). Optionally, the consecutive nucleotides comprise a sequence present in transcript F' (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) that is not present in transcript F (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA). The readthrough portion in transcript F contains a thymine insertion present in the HSD17B13 rs72613567 variant gene, while the readthrough portion in transcript F' does not contain this thymine insertion.

[0179] In yet another embodiment, one or more primers or probes specifically hybridize to a region spanning the boundary between exon 5 and exon 7 in transcript C (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA). It is understood that such primers or probes will be designed to hybridize to a sufficient number of nucleotides in exon 5 and exon 7 to distinguish them from other features in the HSD17B13 transcript (e.g., the boundaries between exon 5 and exon 6 or exon 6 and exon 7 in other HSD17B13 transcripts).

[0180] Certain methods use probes and primers (described in more detail elsewhere herein) of sufficient nucleotide length to bind to a target DNA sequence and specifically detect and / or identify a polynucleotide containing the HSD17B13 rs72613567 variant, or a specific HSD17B13 RNA or mRNA transcript, or a cDNA derived therefrom. To achieve this result, the hybridization or reaction conditions can be determined by the practitioner. The lengths can be any length sufficient to be useful in the selected detection method. Such probes and primers can specifically hybridize to the target sequence under highly stringent hybridization conditions. The probes and primers can have consecutive nucleotides that show complete DNA sequence identity with the target sequence, or probes that differ from the target DNA sequence but retain the ability to specifically detect and / or identify the target DNA sequence can be designed using conventional methods. Thus, probes and primers can have about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the target polynucleotide. In some embodiments, probes and primers can have about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the target polynucleotide. In some embodiments, probes and primers can have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity to the target polynucleotide.

[0181] Specific primers can be used to amplify the HSD17B13 rs72613567 variant gene and / or specific HSD17B13 RNA or mRNA transcripts to generate amplicons that can be used as "specific probes" or that can themselves be detected to identify the HSD17B13 rs72613567 variant gene or determine the level of specific HSD17B13 RNA or mRNA transcripts in a biological sample. The HSD17B13 variant gene can be used to represent a genomic nucleic acid sequence that includes a position corresponding to residue 12666 in SEQ ID NO:2 (a thymine insertion relative to the wild-type genomic locus shown in SEQ ID NO:1 (i.e., inserted between positions 12665 ​​and 12666 in SEQ ID NO:1)). When a probe hybridizes to a polynucleotide in a biological sample under conditions that allow the probe to bind to the sample, this binding can be detected, i.e., indicating the presence of the HSD17B13 rs72613567 variant gene or the presence or level of a specific HSD17B13 RNA or mRNA transcript in the biological sample. Identifying the bound probe in this manner has been described. A specific probe may contain a sequence that is at least about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% identical (about 90% to about 95%, or about 95% to about 100% identical) to (or complementary to) a specific region of the HSD17B13 gene, HSD17B13 RNA or mRNA transcript, or HSD17B13 cDNA derived therefrom.

[0182] To determine whether a nucleic acid molecule in a biological sample contains a thymine insertion at residue 12666 in the HSD17B13 rs72613567 variant gene (e.g., SEQ ID NO: 2) (i.e., whether it contains a thymine insertion between residues 12665 ​​and 12666 in the wild-type HSD17B13 locus (SEQ ID NO: 1)), the biological sample may be subjected to polynucleotide amplification using a primer pair comprising a first primer derived from the 5' flanking sequence adjacent to the thymine insertion and a second primer derived from the 3' flanking sequence adjacent to the thymine insertion to produce an amplicon diagnostic for the presence of a thymine insertion at residue 12666 in the HSD17B13 rs72613567 variant gene (SEQ ID NO: 2) (i.e., a thymine insertion between residues 12665 ​​and 12666 in the wild-type HSD17B13 gene (SEQ ID NO: 1)). In some cases, the length of the amplicon can range from the combined 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 up to the limit of the amplification reaction, i.e., approximately 20,000 nucleotide base pairs. Optionally, the primer pair flanks the region containing the thymine insertion and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides on each side of the thymine.

[0183] PCR primer pairs can be obtained from known sequences using computer programs designed to obtain primer pairs, such as the PCR primer analysis tool 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, primers can be manually identified by visual inspection of the sequence using known guidelines.

[0184] As described in more detail below, any conventional nucleic acid hybridization method, nucleic acid amplification method, or nucleic acid sequencing method can be used to specifically detect the presence of the HSD17B13 rs72613567 variant locus and / or the level of a specific HSD17B13 RNA or mRNA transcript. By "specifically detect," it is intended that the polynucleotide can be used either as a primer to amplify a region of an HSD17B13 polynucleotide, or as a probe that hybridizes under stringent conditions to a polynucleotide comprising the HSD17B13 rs72613567 variant gene or a specific HSD17B13 transcript, particularly transcripts C, D, E, F, G, or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA).

[0185] A variety of techniques are available in the art, including, for example, nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification. Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.

[0186] Other methods include nucleic acid hybridization methods other than sequencing, including the use of labeled primers or probes on purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid 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 nucleotide sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0187] For example, any method can be used to detect unamplified or amplified polynucleotides, including hybridization protection assays (HPA), quantitative assessment of the amplification process in real time, and any method can be used to determine the amount of target sequence initially present in a sample (although this is not real-time amplification-based).

[0188] Also provided are methods for identifying nucleic acid molecules that do not necessarily require amplification of the sequence, such as those based on known methods such as Southern (DNA:DNA) blot hybridization of chromosomal material, in situ hybridization (ISH), and fluorescent in situ hybridization (FISH). Specific nucleotide sequences can be detected using Southern blot techniques. In such methods, nucleic acids extracted from a sample are fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter.

[0189] Examples of suitable quantitative assays include fluorescence in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization to immobilized probe(s), INVADER® probe, TAQMAN® Molecular Beacon probe, or ECLIPSE™ probe techniques. Traditional assays for screening for targeted modifications, such as long-range PCR, Southern blotting, or Sanger sequencing, can also be used. Next-generation sequencing (NGS) can also be used for screening. Next-generation sequencing can also be referred to as "NGS," "massively parallel sequencing," or "high-throughput sequencing."

[0190] In hybridization techniques, stringent conditions can be used to make probe or primer specifically hybridize to its target.In some embodiments, under stringent conditions, polynucleotide primer or probe will hybridize to its target sequence (for example, variant HSD17B13 gene, variant HSD17B13 RNA or cDNA corresponding to its RNA, or variant HSD17B13 mRNA or cDNA corresponding to its mRNA) to a detectably greater extent (at least 2-fold, at least 3-fold, at least 4-fold, or greater than background (including more than 10-fold greater than background)) than other sequences (for example, corresponding wild-type HSD17B13 gene, wild-type HSD17B13 RNA or cDNA corresponding to its RNA, or cDNA corresponding to wild-type HSD17B13 mRNA or mRNA).In some embodiments, under stringent conditions, polynucleotide primer or probe will hybridize to its target sequence to a detectably greater extent (at least 2-fold) than other sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably more (at least 3-fold) than other sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably more (at least 4-fold) than other sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably more (more than 10-fold over background) than other sequences. Stringent conditions are sequence-dependent and will vary depending on the circumstances.

[0191] Suitable stringent conditions for DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C followed by a 2x SSC wash at 50°C, are known and 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 are salt concentrations of less than about 1.5M Na ion, typically about 0.01-1.0M Na ion (or other salt), at pH 7.0-8.3, and temperatures 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., greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. Exemplary low-stringency conditions include hybridization at 37°C in a buffer containing 30-35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50-55°C in 1xSSC to 2xSSC (20xSSC = 3.0 M NaCl / 0.3 M trisodium citrate). Exemplary medium-stringency conditions include hybridization at 37°C in 40-45% formamide, 1.0 M NaCl, and 1% SDS, followed by washing at 55-60°C in 0.5xSSC to 1xSSC. Exemplary high-stringency conditions include hybridization at 37°C in 50% formamide, 1 M NaCl, and 1% SDS, followed by washing at 60-65°C in 0.1xSSC. 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 washing will be at least long enough to allow equilibrium to be reached.

[0192] The present disclosure provides methods for detecting the presence of HSD17B13 isoform D in a human subject, the methods comprising or consisting of performing an assay on a biological sample taken from the human subject, whereby the presence of HSD17B13 isoform D in the biological sample is determined. In some embodiments, HSD17B13 isoform D comprises or consists of an amino acid sequence that is 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 SEQ ID NO: 42. In some embodiments, the assay comprises sequencing.

[0193] The present disclosure provides methods (including, for example, protein sequencing and immunoassays) for detecting the presence or quantitating the level of a variant HSD17B13 polypeptide in a biological sample. In some embodiments, a method for detecting the presence of a variant HSD17B13 polypeptide in a human subject comprises performing an assay on a biological sample obtained from the human subject to detect the presence of a variant HSD17B13 polypeptide in the biological sample.

[0194] Illustrative, non-limiting examples of protein sequencing techniques include, but are not limited to, mass spectrometry and Edman degradation. Illustrative examples of immunoassays include, but are not limited to, immunoprecipitation, Western blot, immunohistochemistry, ELISA, immunocytochemistry, flow cytometry, and immuno-PCR. Using various known techniques, detectably labeled (e.g., calorimetrically, fluorescently, chemiluminescently, or radioactively) polyclonal or monoclonal antibodies are suitable for use in immunoassays. With respect to immunoassays, variant HSD17B13 isoforms differ in size from HSD17B13 isoforms corresponding to the wild-type state, and therefore migrate at different molecular weights on a protein gel. Therefore, by using the same antibody, HSD17B13 isoforms corresponding to the wild-type state can be distinguished from variant HSD17B13 isoforms, for example, in a Western blot assay.

[0195] In some embodiments, the detected HSD17B13 isoform is not translocated from the cell membrane. In some embodiments, the HSD17B13 isoform is a membrane-associated protein. Such association can aid in the processing of certain biological samples (i.e., the collection of membrane-prepared samples).

[0196] The present disclosure also provides kits for making the compositions of the present disclosure and for utilizing the methods described herein. The kits described herein can include one or more assays for detecting one or more genetic variants in a subject's sample.

[0197] In some embodiments, the compositions and methods described above are used in kits for identifying HSD17B13 variants in humans. In some embodiments, a basic kit can include a container containing at least one pair of oligonucleotide primers or probes (e.g., variance-specific probes or variance-specific primers) for hybridizing to any of the nucleic acid molecules disclosed herein. The kit can also optionally include instructions for use. The kit can also include other optional kit components, such as an allelic ladder for each locus to be amplified, a sufficient amount of amplification enzyme, an amplification buffer to facilitate amplification, a divalent cation solution to enhance enzyme activity, dNTPs for chain extension during amplification, a loading solution to prepare amplified material for electrophoresis, genomic DNA as a template control, size markers to ensure material migrates as expected on the separation medium, and protocols and manuals to instruct the user and limit errors during use.

[0198] In some embodiments, any of the kits disclosed herein may further include any one or more of a nucleotide ladder, a protocol, an enzyme (such as an enzyme used in amplification, such as polymerase chain reaction (PCR)), dNTPs, a buffer, a salt or salts, and a control nucleic acid sample. In some embodiments, any of the kits disclosed herein may further include any one or more of a detectable label, products and reagents necessary to perform an annealing reaction, and instructions. In some embodiments, the kit may include one or more of the primers or probes disclosed herein. For example, the kit may include one or more probes that hybridize to one or more of the disclosed genetic variants. In some embodiments, the kit may include one of the disclosed cells or cell lines. The kit may further include medium for cell culture.

[0199] The present disclosure provides a method for determining the susceptibility or risk of a subject to develop liver disease (e.g., chronic liver disease), or for diagnosing a subject for liver disease (e.g., fatty liver disease, NAFLD, or simple fatty liver disease) or the risk of developing liver disease. The subject can be any organism (e.g., including humans, non-human mammals, rodents, mice, or rats). Such methods can comprise or consist of, for example, detecting the presence of the HSD17B13 rs72613567 variant gene in a biological sample comprising genomic DNA, detecting the presence or level of any one of HSD17B13 transcripts C, D, F, G and H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) in a biological sample comprising RNA or cDNA derived from RNA, or comprising cDNA derived from mRNA or mRNA, particularly D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or detecting the presence or level of any one of HSD17B13 isoforms C, D, F, G or H, particularly D, in a biological sample comprising protein. It will be appreciated that gene sequences within a population, and the RNAs, mRNAs and proteins encoded by those genes, may vary due to polymorphisms, such as single nucleotide polymorphisms. The sequences shown herein for the HSD17B13 gene and each HSD17B13 transcript and HSD17B13 isoform are merely exemplary sequences for the HSD17B13 gene and each HSD17B13 transcript and HSD17B13 isoform. Other sequences for the HSD17B13 gene, each HSD17B13 transcript and HSD17B13 isoform are also possible.

[0200] In any of the methods or uses described herein, the liver disease can be chronic liver disease, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple fatty liver disease, steatohepatitis, fibrosis, or non-alcoholic steatohepatitis (NASH). In some embodiments, the liver disease is fatty liver disease, NAFLD, or simple fatty liver disease. In some embodiments, the liver disease is chronic liver disease. In some embodiments, the liver disease is fatty liver disease. In some embodiments, the liver disease is NAFLD. In some embodiments, the liver disease is alcoholic fatty liver disease. In some embodiments, the liver disease is fibrosis. In some embodiments, the liver disease is cirrhosis. In some embodiments, the liver disease is viral hepatitis. In some embodiments, the liver disease is NASH. In some embodiments, the liver disease is hepatocellular carcinoma. In some embodiments, the liver disease is simple fatty liver disease. In some embodiments, the liver disease is steatohepatitis. In some embodiments, the liver disease is fibrosis, NASH, or cirrhosis.

[0201] Liver diseases, such as chronic liver disease, include liver diseases lasting for over six months and may include, for example, liver diseases involving progressive destruction and regeneration of the liver parenchyma, which may lead to fibrosis and cirrhosis. Liver pathologies encompassed by chronic liver disease include, for example, inflammation (e.g., chronic hepatitis), cirrhosis, and hepatocellular carcinoma. Types of chronic liver disease are disclosed elsewhere herein and include, for example, fatty liver disease, viral hepatitis, nonalcoholic fatty liver disease, alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma. Symptoms and signs of chronic liver disease are known and may include, for example, an enlarged liver, fatigue, pain in the right upper abdomen, abdominal distension (ascites), enlarged blood vessels just below the surface of the skin, chest enlargement in men, enlarged spleen, palmar erythema, and yellowing of the skin and eyes (jaundice). Testing for chronic liver disease may involve blood tests, liver imaging, and liver biopsy. If a subject has at least one known risk factor (e.g., a genetic factor such as a pathogenic mutation), the individual is at increased risk of chronic liver disease, and individuals with the risk factor are at a statistically significantly higher risk of developing the disease than individuals without the risk factor. Risk factors for chronic liver disease are also well known, and can include, for example, excessive alcohol consumption, obesity, high cholesterol, high blood triglycerides, polycystic ovary syndrome, sleep apnea, type 2 diabetes, underactive thyroid gland (hypothyroidism), underactive pituitary gland (hypopituitarism), and metabolic syndrome (including elevated blood lipids).

[0202] The present disclosure provides a method for determining a human subject's susceptibility or risk for developing liver disease, comprising: a) performing an assay on a biological sample from the human subject, wherein the assay determines whether a thymine is present between positions 12665 ​​and 12666 of SEQ ID NO: 1 in a wild-type HSD17B13 gene or a thymine is present at position 12666 of SEQ ID NO: 2 in a variant HSD17B13 gene; and b) determining whether a thymine is present between positions 12665 ​​and 12666 of SEQ ID NO: 1 in a wild-type HSD17B13 gene or a thymine is present at position 12666 of SEQ ID NO: 2 in a variant HSD17B13 gene. or classifying the human subject as having a reduced risk of developing liver disease if a thymine is present in the HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2, or as having an increased risk of developing liver disease if a thymine is not inserted between positions 12665 ​​and 12666 of SEQ ID NO: 1 in the HSD17B13 gene, or if a thymine is absent in the HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2. In some embodiments, the liver disease is a chronic liver disease. In some embodiments, the liver disease is selected from the group consisting of fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple steatosis, steatohepatitis, fibrosis, and non-alcoholic steatohepatitis (NASH).In some embodiments, the assay comprises, or consists of: i) contacting the biological sample with a primer that hybridizes to a region of the HSD17B13 gene that is within 50 nucleotides of positions in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or position 12666 of SEQ ID NO:2; ii) extending the primer to at least pass through positions in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or position 12666 of SEQ ID NO:2; and iii) determining whether the extension product of the primer contains a thymine inserted between positions 12665 ​​and 12666 of SEQ ID NO:1 in the wild-type HSD17B13 gene or a thymine present at position 12666 of SEQ ID NO:2 in the variant HSD17B13 gene. In some embodiments, the assay comprises or consists of contacting the biological sample with a primer or probe that specifically hybridizes under stringent conditions to a variant HSD17B13 gene having a thymine at position 12666 of SEQ ID NO:2, but does not hybridize to the corresponding wild-type HSD17B13 gene, and determining whether hybridization has occurred. In some embodiments, the variant HSD17B13 gene is detected by sequencing. In some embodiments, the method further comprises determining whether the human subject is homozygous for the variant HSD17B13 gene.

[0203] In some embodiments, a method of the present disclosure comprises or consists of detecting the presence of the HSD17B13 rs72613567 variant gene in a biological sample containing genomic DNA. Such a method can comprise or consist of: (a) performing an assay on a biological sample obtained from a subject, the biological sample containing the HSD17B13 gene, to determine the type of nucleotide occupying a position in the HSD17B13 gene corresponding to position 12666 or positions 12666 and 12667 of SEQ ID NO:2; and (b) classifying the subject as having a reduced risk of developing liver disease if the position corresponding to position 12666 of SEQ ID NO:2 is occupied by thymine, or if positions 12666 and 12667 of SEQ ID NO:2 are occupied by thymine. Alternatively, the subject can be classified as having an increased risk of developing liver disease if the positions are not occupied by thymine. Similarly, such methods can include performing an assay on a biological sample to determine the type of nucleotide occupying positions in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1. If a thymine is inserted between positions 12665 ​​and 12666 of SEQ ID NO: 1 in the wild-type HSD17B13 gene, the subject can be classified as having a reduced risk of developing liver disease. Alternatively, if a thymine insertion is not found between positions 12665 ​​and 12666 of SEQ ID NO: 1 in the wild-type HSD17B13 gene, the subject can be classified as having an increased risk of developing chronic liver disease.

[0204] Any assay can be used to determine the type of nucleotide occupying a position in the HSD17B13 gene corresponding to positions 12666 or 12666 and 12667 of SEQ ID NO:2 (or positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1). By way of example, the assay can comprise or consist of sequencing a portion of the HSD17B13 gene including a position corresponding to positions 12666 or 12666 and 12667 of SEQ ID NO:2. The sequencing may comprise or consist of: i) contacting the biological sample with a primer that hybridizes to a segment of the HSD17B13 gene that is adjacent to a position in the HSD17B13 gene corresponding to position 12666 or positions 12666 and 12667 of SEQ ID NO:2; ii) extending the primer so that it passes through at least a position in the HSD17B13 gene that corresponds to position 12666 or positions 12666 and 12667 of SEQ ID NO:2; and iii) determining which of the extension products of the primer is at a position in the HSD17B13 gene that corresponds to position 12666 or positions 12666 and 12667 of SEQ ID NO:2. As used herein, "close to" means within about 50 nucleotides, within about 45 nucleotides, within about 40 nucleotides, within about 35 nucleotides, within about 30 nucleotides, within about 25 nucleotides, within about 20 nucleotides, within about 15 nucleotides, within about 10 nucleotides, or within about 5 nucleotides of the particular position indicated.

[0205] As another example, the above assay can include or consist of contacting a biological sample with a primer or probe (such as a variation-specific primer or variation-specific probe) that specifically hybridizes to the variant HSD17B13 rs72613567 gene and does not hybridize to the corresponding wild-type HSD17B13 gene under stringent conditions, and determining whether hybridization has occurred.

[0206] The present disclosure provides a method for determining a human subject's susceptibility or risk for developing liver disease, comprising, or consisting of, a) performing an assay on a biological sample obtained from the human subject, wherein the assay determines the presence of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA) in the biological sample; and b) classifying the human subject as having a reduced risk for developing liver disease if HSD17B13 transcript D is present in the biological sample, or as having an increased risk for developing liver disease if HSD17B13 transcript D is absent from the biological sample. In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 (at least about 90% identical) to SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the HSD17B13 transcript D is RNA and its cDNA comprises or consists of SEQ ID NO: 6, or comprises or consists of SEQ ID NO: 24, or the HSD17B13 transcript D is mRNA and its cDNA comprises or consists of SEQ ID NO: 15, or comprises or consists of SEQ ID NO: 33.In some embodiments, the assay determines the expression level of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) in a biological sample, where an elevated expression level of HSD17B13 transcript D compared to a control sample from a control human subject homozygous for the wild-type HSD17B13 allele indicates a decreased risk of developing liver disease, and a consistent or decreased expression level of HSD17B13 transcript D compared to the control sample indicates an increased risk of developing liver disease. In some embodiments, the liver disease is a chronic liver disease. In some embodiments, the liver disease is selected from the group consisting of fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple steatosis, steatohepatitis, fibrosis, and non-alcoholic steatohepatitis (NASH). In some embodiments, the assay comprises or consists of contacting the biological sample with one or more primers or probes that specifically hybridize to a nucleic acid sequence of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA), or its complement, and determining whether hybridization has occurred.In some embodiments, the method comprises: i) a nucleotide sequence that is 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 (i.e., at least about 90% identical) to the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or a complement thereof; ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA); and / or iii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D (RNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA). The method further comprises specifically detecting transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) by using a nucleic acid molecule comprising or consisting of about 5 to a maximum of about 50 nucleotides that comprises or consists of a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of product D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA). In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 (i.e., at least about 90% identical) to SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the one or more primers or probes specifically hybridize to SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, and / or SEQ ID NO: 33. In some embodiments, the assay comprises reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). In some embodiments, the assay comprises sequencing.

[0207] In some embodiments, the methods of the disclosure comprise, or consist of, a) performing an assay on a biological sample obtained from a subject, the biological sample comprising RNA or cDNA derived from RNA, or comprising mRNA or cDNA derived from mRNA, wherein the assay determines the presence of transcript C, D, F, G, or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) in the biological sample; and b) classifying the subject as having a reduced risk of developing liver disease if transcript C, D, F, G, or H is present in the biological sample. Such assays can detect, for example, regions or combinations of regions present in one or more of transcripts C, D, F, G, and H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) that are absent from transcripts A and B (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or that are absent from transcripts A, B, and E (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or that are absent from transcripts A, B, E, and F' (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) (e.g., regions or combinations of regions that are unique to transcripts C, D, F, G, and H). Such regions can be readily identified by comparing the sequences of transcripts A-H and are described in further detail elsewhere herein. Alternatively, if transcripts C, D, F, G or H are not present in the biological sample, the subject can be classified as being at increased risk for developing liver disease.In a specific example, the assay can determine the expression level of transcripts C, D, F, G, or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), particularly transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) in a biological sample, where an increased expression level of transcripts C, D, F, G, or H, particularly transcript D, in the biological sample compared to a control sample obtained from a control subject homozygous for the wild-type HSD17B13 allele indicates a decreased risk of developing liver disease. Alternatively, a decreased or unchanged expression level of transcripts C, D, F, G, or H, particularly transcript D, in the biological sample compared to a control sample obtained from a control subject homozygous for the wild-type HSD17B13 allele indicates an increased risk of developing liver disease.In another embodiment, the assay is carried out by measuring the expression level of transcript C, D, F, G or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), in particular transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in a biological sample, by measuring the expression level of transcript A, B or E (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or transcript A, B, E or F' (RNA or cDNA derived from RNA or RNA), in particular transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in a biological sample. This may include comparing the expression of transcripts C, D, F, G, or H, particularly transcript D, to the expression of transcripts A, B, or E, or transcripts A, B, E, or F', particularly transcript A, compared to that in a control sample obtained from a control subject homozygous for the wild-type HSD17B13 allele, indicating a reduced risk of developing liver disease. Alternatively, a lower or unchanged ratio of the expression of transcripts C, D, F, G, or H, particularly transcript D, to the expression of transcripts A, B, or E, or transcripts A, B, E, and F', particularly transcript A, compared to that in a control sample obtained from a control subject homozygous for the wild-type HSD17B13 allele indicates an increased risk of developing liver disease.

[0208] In some methods for detecting the presence or level of any one of transcripts C, D, F, G or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), in particular D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), the assay comprises detecting transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), transcript G (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), The method may include or consist of contacting the biological sample with one or more primers or probes (e.g., variation-specific primers or variation-specific probes) that specifically hybridize to a region spanning the boundary between exon 6 and exon 7 of transcript H (cDNA derived from RNA, preferably cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) (i.e., a region containing an additional guanine at the 3' end of exon 6 (exon 6 of other HSD17B13 transcripts does not have an additional guanine)), and determining whether hybridization has occurred. Additionally or alternatively, the assay may include contacting the biological sample with one or more primers or probes that specifically hybridize to a region within the read-through into intron 6 of transcript F (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) or to a region spanning the boundary between the read-through into intron 6 of transcript F (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) and the remainder of exon 6, and determining whether hybridization has occurred.Additionally or alternatively, the assay can include contacting the biological sample with one or more primers or probes that specifically hybridize to a region spanning the boundary between exon 5 and exon 7 of transcript C (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA), and determining whether hybridization has occurred.

[0209] Other assays that can be used in the methods disclosed herein include, for example, reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). Yet another assay that can be used in the methods disclosed herein includes, for example, RNA sequencing (RNA-Seq) followed by determining the presence and amount of transcripts C, D, F, G, or H, particularly transcript D, in a biological sample.

[0210] Other methods may include detecting the presence or level of any one of HSD17B13 transcripts A, B, and E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) or transcripts A, B, E, and F' (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) in a biological sample. Such a method may comprise, or consist of, a) performing an assay on a biological sample obtained from the subject, which assay determines the presence in the biological sample of transcript A, B, or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or transcript A, B, E, or F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA); and b) classifying the subject as having an increased risk of developing liver disease if transcript A, B, or E, or transcript A, B, E, or F' is present in the biological sample. Such assays can detect, for example, a region or combination of regions that is present in one or more of transcripts A, B, or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or transcripts A, B, E, or F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), but is not present in transcripts C, D, F, G, and H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) (e.g., a region or combination of regions that is unique to transcript A, B, or E, or one or more of transcripts A, B, E, or F').Such regions can be readily identified by comparing the sequences of transcripts A-H, as described in more detail elsewhere herein. Alternatively, the absence of transcript A, B, or E, or transcript A, B, E, or F' in a biological sample can classify the subject as having a reduced risk of developing liver disease. In a specific example, the assay can determine the expression level of transcript A, B, or E (RNA or cDNA derived from RNA and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) or transcript A, B, E, or F' (RNA or cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) in a biological sample, where an elevated expression level of transcript A, B, or E, or transcript A, B, E, or F' in a biological sample compared to a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates an elevated risk of developing liver disease. Alternatively, a reduced or unchanged expression level of transcript A, B or E, or transcript A, B, E or F' in the biological sample compared to a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates a reduced risk of developing liver disease.In another embodiment, the assay may comprise determining in a biological sample the expression level of transcript A, B or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or transcript A, B, E or F' (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), relative to transcript C, D, F, G or H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), particularly transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), wherein the ratio of expression of transcript A, B or E, or transcript A, B, E or F' to expression of transcript C, D, F, G or H, particularly transcript D, is A higher ratio compared to that in a control sample obtained from a control subject homozygous for the rs72613567 variant allele indicates an increased risk of developing liver disease. Alternatively, a lower or unchanged ratio of the expression of transcripts A, B, or E, or transcripts A, B, E, or F' to that of transcripts C, D, F, G, or H, particularly transcript D, compared to that in a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates a decreased risk of developing liver disease.

[0211] In some methods for detecting the presence or level of any one of transcripts A, B, or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or transcripts A, B, E, or F' (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), the assay involves analyzing a region within exon 3', exon 3, and exon 3' in transcript E (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), to distinguish transcript E from transcripts A, B, C, D, F, and G. The method may include or consist of contacting the biological sample with one or more primers or probes (such as a variation-specific primer or a variation-specific probe) that specifically hybridize to a region spanning the boundary between exons 6 and 7 in transcript E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript E from transcript H, and determining whether hybridization has occurred.Additionally, or alternatively, the assay may comprise or consist of contacting the biological sample with one or more primers or probes (such as variation-specific primers or variation-specific probes) that specifically hybridize to a region spanning the boundary between exon 1 and exon 3 of transcript B (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript B from transcripts A, C, D, E, F, and H; further contacting the biological sample with one or more primers or probes that specifically hybridize to a region spanning the boundary between exon 6 and exon 7 in transcript B (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript B from transcript G; and determining whether hybridization has occurred. Additionally or alternatively, the assay may comprise or consist of contacting the biological sample with one or more primers or probes (such as variation-specific primers or variation-specific probes) that specifically hybridize to a region spanning the boundary between exon 6 and exon 7 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript A from transcripts D, F, G, and H; further contacting the biological sample with one or more primers or probes (such as variation-specific primers or variation-specific probes) that specifically hybridize to a region within exon 6, a region spanning the boundary between exon 5 and exon 6, or a region spanning the boundary between exon 6 and exon 7 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript A from transcripts D, F, G, and H; and determining whether hybridization has occurred.Optionally, the assay can further include contacting the biological sample with one or more primers or probes (e.g., variation-specific primers or variation-specific probes) that specifically hybridize to a region within exon 2, a region spanning the boundary between exon 1 and exon 2, or a region spanning the boundary between exon 2 and exon 3 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), to distinguish transcript A from transcripts B and H; and further contacting the biological sample with one or more primers or probes (e.g., variation-specific primers or variation-specific probes) that specifically hybridize to a region spanning the boundary between exon 3 and exon 4 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), to distinguish transcript A from transcripts E and H; and determining whether hybridization has occurred.

[0212] Other assays that can be used in the methods disclosed herein include, for example, reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). Yet another assay that can be used in the methods disclosed herein includes, for example, determining the presence and amount of transcript A, B, or E, or transcript A, B, E, or F' in a biological sample after RNA sequencing (RNA-Seq).

[0213] The present disclosure provides methods for determining a human subject's susceptibility or risk for developing liver disease, comprising or consisting of: a) detecting the presence of HSD17B13 isoform D in a biological sample obtained from the human subject; and b) classifying the human subject as having a reduced risk for developing liver disease if HSD17B13 isoform D is detected in the biological sample, or classifying the human subject as having a reduced risk for developing liver disease if HSD17B13 isoform D is not detected in the biological sample. In some embodiments, HSD17B13 isoform D comprises or consists of an amino acid sequence 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 (i.e., at least about 90% identical) to SEQ ID NO: 42. In some embodiments, the liver disease is chronic liver disease. In some embodiments, the liver disease is selected from the group consisting of fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple steatosis, steatohepatitis, fibrosis, and non-alcoholic steatohepatitis (NASH). In some embodiments, the detection comprises sequencing.

[0214] In some embodiments, the methods of the disclosure comprise or consist of detecting the presence or level of any one of HSD17B13 isoforms C, D, F, G, or H, particularly D, in a biological sample containing the protein. Such epitopes can be readily identified by comparing the sequences of isoforms A through H, and are described in further detail elsewhere herein. Alternatively, the absence of isoforms C, D, F, G, or H in the biological sample can classify the subject as being at increased risk for developing liver disease.

[0215] In some embodiments, the detection procedure determines the expression level of isoforms C, D, F, G, or H in the biological sample, and an elevated expression level of isoforms C, D, F, G, or H relative to a control sample from a control subject homozygous for the wild-type HSD17B13 allele indicates a reduced risk of developing liver disease. Alternatively, a decreased or unchanged expression level of isoforms C, D, F, G, or H relative to a control sample from a control subject homozygous for the wild-type HSD17B13 allele indicates an elevated risk of developing liver disease.

[0216] In some embodiments, the detection procedure involves determining the expression level of isoforms A, B, or E, or isoforms A, B, E, or F' in the biological sample, where an elevated expression level of isoforms A, B, or E, or isoforms A, B, E, or F' compared to a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates an elevated risk of developing liver disease. Alternatively, a decreased or unchanged expression level of isoforms A, B, or E, or isoforms A, B, E, or F' compared to a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates a decreased risk of developing liver disease.

[0217] The present disclosure provides methods for determining a human subject's risk of progressing to a more clinically advanced stage of fatty liver disease. The present disclosure provides a method for determining the risk of a human subject progressing to a clinically more advanced stage of fatty liver disease, comprising: a) performing an assay on a biological sample from the human subject, wherein the assay determines whether a thymine is present in a wild-type HSD17B13 gene at a position corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1 or a variant HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2; and b) determining whether a thymine is present in a wild-type HSD17B13 gene at a position corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1 or a variant HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2. classifying the human subject as having a reduced risk of progressing to a clinically more advanced stage of fatty liver disease if a thymine is present in the HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2, or classifying the human subject as having an increased risk of progressing to a clinically more advanced stage of fatty liver disease if there is no thymine inserted between positions 12665 ​​and 12666 of SEQ ID NO: 1 in the HSD17B13 gene or if there is no thymine present in the HSD17B13 gene at a position corresponding to position 12666 of SEQ ID NO: 2. In some embodiments, the assay comprises, or consists of: i) contacting the biological sample with a primer that hybridizes to a region of the HSD17B13 gene that is within 50 nucleotides of positions in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or position 12666 of SEQ ID NO:2; ii) extending the primer to at least pass through positions in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1 or position 12666 of SEQ ID NO:2; and iii) determining whether the extension product of the primer contains a thymine inserted between positions 12665 ​​and 12666 of SEQ ID NO:1 in the wild-type HSD17B13 gene or a thymine present at position 12666 of SEQ ID NO:2 in the variant HSD17B13 gene.In some embodiments, the assay comprises or consists of contacting the biological sample with a primer or probe that specifically hybridizes under stringent conditions to a variant HSD17B13 gene having a thymine at position 12666 of SEQ ID NO:2, but does not specifically hybridize to the corresponding wild-type HSD17B13 gene, and determining whether hybridization has occurred. In some embodiments, the variant HSD17B13 gene is detected by sequencing. In some embodiments, the method further comprises determining whether the human subject is homozygous for the variant HSD17B13 gene.

[0218] In some embodiments, the methods of the disclosure comprise or consist of: a) performing an assay on a biological sample obtained from a subject comprising the HSD17B13 gene, wherein the assay determines the type of nucleotide occupying a position in the HSD17B13 gene corresponding to position 12666 or positions 12666 and 12667 of SEQ ID NO:2; and b) classifying the subject as having a reduced risk of progressing to a more clinically advanced stage of liver disease (e.g., the risk of progressing from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma) if the position corresponding to position 12666 of SEQ ID NO:2 is occupied by thymine, or if positions 12666 and 12667 of SEQ ID NO:2 are occupied by thymine. Alternatively, if the position is not occupied by thymine, the subject can be classified as having an increased risk of progressing to a clinically more advanced stage of liver disease (e.g., the risk of histopathologically progressing from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma). Similarly, such a method can comprise or consist of performing an assay on a biological sample to determine the type of nucleotide occupying positions in the HSD17B13 gene corresponding to positions 12665 ​​and 12666 of SEQ ID NO: 1. If a thymine is inserted between positions 12665 ​​and 12666 of SEQ ID NO: 1 in the wild-type HSD17B13 gene, the subject can be classified as having a decreased risk of progressing to a clinically more advanced stage of liver disease (e.g., the risk of histopathologically progressing from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma). Alternatively, if no thymine insertion is found between positions 12665 ​​and 12666 of SEQ ID NO: 1 in the wild-type HSD17B13 gene, the subject can be classified as being at increased risk of progressing to a more clinically advanced stage of liver disease (e.g., histopathologically, the risk of progressing from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma).

[0219] In some embodiments, if a subject is determined to have HSD17B13 protein isoform A, B, E, or F', or transcript A, B, E, or F', the subject is at an increased risk of developing fibrosis, which may manifest as late-stage NASH. In contrast, if a subject is determined to have HSD17B13 protein isoform C, D, F, G, or H, or transcript C, D, F, G, or H, the subject is at a reduced risk of developing fibrosis. In some embodiments, subjects at or suspected of being at an increased risk of developing fibrosis can also be examined for histopathological features of NASH (e.g., including lobular inflammation and hepatocellular ballooning). Subjects with HSD17B13 protein isoform C, D, F, G, or H, or transcript C, D, F, G, or H, are at a reduced risk of developing lobular inflammation and hepatocellular ballooning.

[0220] Any assay (such as those described herein) for determining the identity of the nucleotide occupying a position in the HSD17B13 gene corresponding to positions 12666 or 12666 and 12667 of SEQ ID NO: 2 (or positions 12665 ​​and 12666 of SEQ ID NO: 1) can be used. Additionally, any assay (such as those described herein) can include or consist of contacting a biological sample with a primer or probe (such as a variation-specific primer or variation-specific probe) that specifically hybridizes to the HSD17B13 rs72613567 variant but does not hybridize to the corresponding wild-type HSD17B13 sequence under stringent conditions, and determining whether hybridization has occurred.

[0221] The present disclosure provides a method for determining a human subject's risk of progressing to a clinically more advanced stage of fatty liver disease, comprising, or consisting of: a) performing an assay on a biological sample obtained from the human subject, wherein the assay determines the presence of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) in the biological sample; and b) classifying the human subject as having a reduced risk of progressing to a clinically more advanced stage of fatty liver disease if HSD17B13 transcript D is present in the biological sample, or as having an increased risk of progressing to a clinically more advanced stage of fatty liver disease if HSD17B13 transcript D is absent from the biological sample. In some embodiments, HSD17B13 transcript D comprises or consists of a nucleotide sequence that is 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 (i.e., at least about 90% identical) to SEQ ID NO: 6, 15, 24, or 33. In some embodiments, the HSD17B13 transcript D is RNA and its cDNA comprises or consists of SEQ ID NO: 6, or comprises or consists of SEQ ID NO: 24, or the HSD17B13 transcript D is mRNA and its cDNA comprises or consists of SEQ ID NO: 15, or comprises or consists of SEQ ID NO: 33.In some embodiments, the assay determines the expression level of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) in a biological sample, wherein an elevated expression level of HSD17B13 transcript D compared to a control sample obtained from a control human subject who is homozygous for the wild-type HSD17B13 allele indicates a reduced risk of progressing to a more clinically advanced stage of fatty liver disease, and a similar or decreased expression level of HSD17B13 transcript D compared to the control sample indicates an increased risk of progressing to a more clinically advanced stage of fatty liver disease. In some embodiments, the assay comprises or consists of contacting the biological sample with one or more primers or probes that specifically hybridize to a nucleic acid sequence of HSD17B13 transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA), or its complement, and determining whether hybridization has occurred.In some embodiments, the method comprises: i) a nucleotide sequence that is 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 (i.e., at least about 90% identical) to the nucleotide sequence of SEQ ID NO: 6, 15, 24, or 33, or a complement thereof; ii) a nucleic acid molecule that specifically hybridizes to exon 2 of transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA); and / or or iii) specifically detecting transcript D (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA) by using a nucleic acid molecule comprising from about 5 nucleotides up to about 50 nucleotides that comprises or consists of a nucleic acid molecule that specifically hybridizes to a region bridging exon 3 and exon 4 of transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA). In some embodiments, the one or more primers or probes specifically hybridize to SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24, and / or SEQ ID NO:33. In some embodiments, the assay comprises reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). In some embodiments, the assay comprises sequencing.

[0222] In some embodiments, the methods of the disclosure comprise, or consist of, performing an assay on a biological sample obtained from a subject, which assay determines the presence in the biological sample of transcript C, D, F, G, or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), in particular transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA); and b) classifying the subject as having a reduced risk of progressing to a clinically more advanced stage of liver disease (e.g., the risk of histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma) if transcript C, D, F, G, or H, in particular transcript D, is present in the biological sample. Alternatively, if transcripts C, D, F, G or H, and particularly transcript D, are not present in the biological sample, the subject can be classified as being at increased risk of progressing to a clinically more advanced stage of liver disease (e.g., histopathological risk of progressing from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma). In a specific example, the assay can determine the expression level of transcripts C, D, F, G or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), particularly transcript D (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in a biological sample, and an increase in the expression level of transcripts C, D, F, G or H, particularly transcript D, in the biological sample compared to a control sample obtained from a control subject homozygous for the wild-type HSD17B13 allele indicates a reduced risk of progression to a clinically more advanced stage of liver disease (e.g., the risk of histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma).Alternatively, a reduced or unchanged expression level of transcripts C, D, F, G or H, particularly transcript D, in a biological sample compared to a control sample obtained from a control subject who is homozygous for the wild-type HSD17B13 allele indicates an increased risk of progression to a more clinically advanced stage of liver disease (e.g., the risk of histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma). In another specific example, the assay of the disclosure is for measuring the expression level of transcript C, D, F, G or H (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), particularly transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in a biological sample, or transcript A, B or E (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), or transcript A, B, E or F' (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in a biological sample. or cDNA derived from RNA), in particular transcript A (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA), wherein a higher ratio of expression of transcript C, D, F, G or H, in particular transcript D, to expression of transcript A, B or E, or transcript A, B, E or F', in particular transcript A, compared to that in a control sample from a control subject homozygous for the wild-type HSD17B13 allele indicates a reduced risk of progression to a more clinically advanced stage of liver disease (e.g., the risk of histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma).Alternatively, a lower or unchanged ratio of expression of transcripts A, B or E, or transcripts A, B, E or F', particularly transcript A, to expression of transcripts C, D, F, G or H, particularly transcript D, indicates an increased risk of progression to a clinically more advanced stage of liver disease (e.g., histopathological risk of progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma).

[0223] In some embodiments, the assay comprises detecting the presence or level of any one of transcripts C, D, F, G or H (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in particular D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), transcript G (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), The method may include, or consist of, contacting the biological sample with one or more primers or probes (e.g., variation-specific primers or variation-specific probes) that specifically hybridize to the region spanning the boundary between exon 6 and exon 7 in transcript H (cDNA derived from NA, preferably cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) or transcript H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) (i.e., the region containing an additional guanine at the 3' end of exon 6 (this additional guanine is not present in exon 6 of other HSD17B13 transcripts)), and determining whether hybridization has occurred. Additionally, or alternatively, the assay may include, or consist of, contacting the biological sample with one or more primers or probes that specifically hybridize to a region within the read-through into intron 6 in transcript F (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or to a region spanning the boundary between the read-through into intron 6 and the remainder of exon 6 in transcript F (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or cDNA derived from RNA or RNA), and determining whether hybridization has occurred.Additionally, or alternatively, the assay can include or consist of contacting the biological sample with one or more primers or probes that specifically hybridize to a region spanning the boundary between exon 5 and exon 7 in transcript C (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably RNA or cDNA derived from RNA), and determining whether hybridization has occurred.

[0224] Other assays that can be used in the methods disclosed herein include, for example, reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). Yet another assay that can be used in the methods disclosed herein includes, for example, RNA sequencing (RNA-Seq) followed by determining the presence and amount of transcripts C, D, F, G, or H, particularly transcript D, in a biological sample.

[0225] In some embodiments, the methods of the disclosure comprise, or consist of, a) performing an assay on a biological sample obtained from the subject, wherein the assay determines the presence of transcript A, B, or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA) or transcript A, B, E, or F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) in the biological sample; and b) classifying the subject as having an increased risk of progressing to a clinically more advanced stage of liver disease (e.g., histopathological risk of progressing from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma) if transcript A, B, or E, or transcript A, B, E, or F' is present in the biological sample. Alternatively, if transcript A, B, or E, or transcript A, B, E, or F' is not present in the biological sample, the subject can be classified as having a reduced risk of progressing to a more clinically advanced stage of liver disease (e.g., histopathological risk of progressing from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma).In a specific example, the assay can determine the expression level of transcript A, B, or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or transcript A, B, E, or F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) in a biological sample, and an elevated expression level of transcript A, B, or E, or transcript A, B, E, or F' in the biological sample compared to a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates an elevated risk of progression to a more clinically advanced stage of liver disease (e.g., histopathological risk of progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma). Alternatively, a reduced or unchanged expression level of transcript A, B or E, or transcript A, B, E or F' in a biological sample compared to a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates a reduced risk of progression to a more clinically advanced stage of liver disease (e.g., the risk of histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma).In another embodiment, the assay may comprise determining in a biological sample the expression level of transcript A, B or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or transcript A, B, E or F' (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), relative to transcript C, D, F, G or H (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), particularly transcript D (cDNA derived from RNA or RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), wherein the ratio of expression of transcript A, B or E, or transcript A, B, E or F' to expression of transcript C, D, F, G or H, particularly transcript D, is A higher ratio compared to control samples from control subjects homozygous for the rs72613567 variant allele indicates an increased risk of progression to more clinically advanced stages of liver disease (e.g., histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma).Alternatively, the ratio of the expression of transcript A, B or E (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) or transcript A, B, E or F' (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to the expression of C, D, F, G or H (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), in particular transcript D (cDNA derived from RNA or RNA and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) is HSD17B13 A lower or unchanged ratio compared to that in control samples from control subjects homozygous for the rs72613567 variant allele indicates a reduced risk of progression to more clinically advanced stages of liver disease (e.g., histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma).

[0226] In some embodiments, to detect the presence or level of any one of transcripts A, B, or E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), or transcripts A, B, E, or F' (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), the assays of the disclosure utilize a region within exon 3', exon 3 and exon 4 in transcript E (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA), such that transcript E is distinguished from transcripts A, B, C, D, F, and G. The method may include or consist of contacting the biological sample with one or more primers or probes (such as a variation-specific primer or a variation-specific probe) that specifically hybridize to a region spanning the boundary of exon 3' or a region spanning the boundary between exon 3' and exon 4, and further contacting the biological sample with one or more primers and probes (such as a variation-specific primer or a variation-specific probe) that specifically hybridize to a region spanning the boundary between exon 6 and exon 7 in transcript E (RNA or cDNA derived from RNA, and / or mRNA or cDNA derived from mRNA, preferably cDNA derived from RNA or RNA), so as to distinguish transcript E from transcript H, and determining whether hybridization has occurred.Additionally, or alternatively, the assay may comprise or consist of contacting the biological sample with one or more primers or probes (such as variation-specific primers or variation-specific probes) that specifically hybridize to a region spanning the boundary between exon 1 and exon 3 of transcript B (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript B from transcripts A, C, D, E, F, and H; further contacting the biological sample with one or more primers or probes that specifically hybridize to a region spanning the boundary between exon 6 and exon 7 in transcript B (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript B from transcript G; and determining whether hybridization has occurred. Additionally or alternatively, the assay may comprise or consist of contacting the biological sample with one or more primers or probes (such as variation-specific primers or variation-specific probes) that specifically hybridize to a region spanning the boundary between exon 6 and exon 7 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript A from transcripts D, F, G, and H; further contacting the biological sample with one or more primers or probes (such as variation-specific primers or variation-specific probes) that specifically hybridize to a region within exon 6, a region spanning the boundary between exon 5 and exon 6, or a region spanning the boundary between exon 6 and exon 7 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript A from transcripts D, F, G, and H; and determining whether hybridization has occurred.Optionally, the assay may further comprise, or alternatively may consist of, contacting the biological sample with one or more primers or probes (such as a variation-specific primer or a variation-specific probe) that specifically hybridize to a region within exon 2, a region spanning the boundary between exons 1 and 2, or a region spanning the boundary between exons 2 and 3 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript A from transcripts B and H; contacting the biological sample with one or more primers or probes (such as a variation-specific primer or a variation-specific probe) that specifically hybridize to a region spanning the boundary between exons 3 and 4 in transcript A (RNA or cDNA derived from RNA, and / or cDNA derived from mRNA or mRNA, preferably cDNA derived from RNA or RNA) to distinguish transcript A from transcripts E and H; and determining whether hybridization has occurred.

[0227] Other assays that can be used in the methods disclosed herein include, for example, reverse transcription polymerase chain reaction (RT-PCR) or quantitative RT-PCR (qRT-PCR). Yet another assay that can be used in the methods disclosed herein includes, for example, determining the presence and amount of transcript A, B, or E, or transcript A, B, E, or F' in a biological sample after RNA sequencing (RNA-Seq).

[0228] The present disclosure provides a method for determining a human subject's risk of progressing to a clinically more advanced stage of fatty liver disease, comprising or consisting of: a) detecting the presence of HSD17B13 isoform D in a biological sample obtained from the human subject; and b) classifying the human subject as having a reduced risk of progressing to a clinically more advanced stage of liver disease if HSD17B13 isoform D is detected in the biological sample. In some embodiments, the HSD17B13 isoform D comprises or consists of an amino acid sequence 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 (i.e., at least about 90% identical) to SEQ ID NO: 42. In some embodiments, the detection step comprises sequencing.

[0229] In some embodiments, the detection procedure involves determining the expression level of isoforms C, D, F, G, or H in the biological sample, and an elevated expression level of isoforms C, D, F, G, or H compared to a control sample obtained from a control human subject homozygous for the wild-type HSD17B13 allele indicates a reduced risk of progression to a clinically more advanced stage of liver disease (e.g., the risk of histopathological progression from simple steatosis to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma). Alternatively, a reduced or unchanged expression level of isoforms C, D, F, G, or H compared to a control sample obtained from a control subject homozygous for the wild-type HSD17B13 allele indicates an elevated risk of progression to a clinically more advanced stage of liver disease (e.g., the risk of histopathological progression from simple steatosis to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma).

[0230] In some embodiments, the detection procedure involves determining the expression level of isoforms A, B, or E, or isoforms A, B, E, or F' in a biological sample, wherein an elevated expression level of isoforms A, B, or E, or isoforms A, B, E, or F' relative to a control sample obtained from a control human subject homozygous for the HSD17B13 rs72613567 variant allele indicates an elevated risk of progression to a more clinically advanced stage of liver disease (e.g., a histopathological risk of progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma). Alternatively, reduced or unchanged expression levels of isoforms A, B or E, or isoforms A, B, E or F' compared to a control sample obtained from a control subject homozygous for the HSD17B13 rs72613567 variant allele indicates a reduced risk of progression to a more clinically advanced stage of liver disease (e.g., the risk of histopathological progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma).

[0231] Any one or more of the methods described herein may be performed in vitro. In any of the methods disclosed herein, a primer or probe may either hybridize to its intended target nucleic acid molecule or specifically hybridize to its intended target nucleic acid molecule. In some embodiments, a primer or probe that specifically hybridizes to a particular target does not hybridize to a wild-type nucleic acid molecule (e.g., SEQ ID NO: 1, or a transcript having functional activity associated with wild-type HSD17B13).

[0232] In any of the methods disclosed herein for classifying a subject as being at increased risk for progression to a clinically more advanced stage of liver disease (e.g., histopathological risk of progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma) or for developing liver disease (e.g., chronic liver disease), the method can further include a therapeutic or prophylactic method. Alternatively, the disclosed methods can further include administering a therapeutic agent to prevent or alleviate one or more symptoms associated with progression to a clinically more advanced stage of liver disease (e.g., progression from simple fatty liver to a clinically more advanced stage of liver disease, or progression from simple fatty liver to one or more of steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma). For example, such treatment can focus on preventing or reducing inflammation or fibrosis. Examples of such therapeutic agents under development include, but are not limited to, obeticholic acid, GS-9674, simtuzumab, GS-4997, NDI-010976, GFT505 / elafibranor, aramchol, cenicriviroc, GR-MD-02, TD139, SHP626, PXS4728A, and RP103-cysteamine tartrate. The present disclosure provides a therapeutic agent selected from the group consisting of the therapeutic agents disclosed above for use in treating, preventing, or ameliorating liver disease in a human subject who is not a carrier of a variant HSD17B13 gene. In one embodiment, the human subject has tested negative for the variant HSD17B13 gene. In one embodiment, the treatment includes determining whether the human subject is a carrier of a variant HSD17B13 gene. In one embodiment, the human patient has been determined to have a variant HSD17B13 gene according to any of the methods described herein. The present disclosure also provides a therapeutic agent selected from the group consisting of the therapeutic agents disclosed above for use in treating, preventing or alleviating fatty liver disease in a human subject determined to be at risk of progressing to a clinically more advanced stage of fatty liver disease according to any of the methods of the present disclosure.

[0233] Various methods are provided for modifying cells through the use of any combination of antisense molecules (such as antisense RNA, siRNA, and shRNA), HSD17B13 protein or fragments thereof, and expression vectors for expressing recombinant HSD17B13 genes or nucleic acids encoding HSD17B13 proteins. The methods can be performed in vitro, ex vivo, or in vivo. Antisense molecules such as antisense RNA, siRNA, and shRNA, HSD17B13 protein or fragments thereof, and expression vectors can be introduced into cells in any form by any means described elsewhere herein, and all or part of them can be introduced simultaneously or sequentially in any combination.

[0234] Antisense molecules can be used to modify the expression of the nucleic acid encoding HSD17B13 gene or HSD17B13 isoform protein.Examples of antisense molecules include antisense RNA, small interfering RNA (siRNA) and short hairpin RNA (shRNA).Such antisense RNA, siRNA or shRNA can be designed to target any region of mRNA.For example, antisense RNA, siRNA or shRNA can be designed to target a region that is unique to one or more of the HSD17B13 transcripts disclosed herein, or a region that is common to one or more of the HSD17B13 transcripts disclosed herein.

[0235] The subject can be, for example, a subject (e.g., a human) who is not a carrier of the HSD17B13 rs72613567 variant (or who is only a heterozygous carrier of the HSD17B13 rs72613567 variant) and who has or is susceptible to developing liver disease.

[0236] Representative embodiments are shown below. Embodiment 1. A nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides of the HSD17B13 gene, wherein a thymine is inserted between the nucleotides at positions corresponding to positions 12665 ​​and 12666 of SEQ ID NO:1.

[0237] Embodiment 2. The nucleic acid molecule of embodiment 1, wherein the consecutive nucleotides are at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequence in SEQ ID NO:2, the sequence including the position corresponding to position 12666 of SEQ ID NO:2.

[0238] Embodiment 3. The nucleic acid molecule of embodiment 1 or 2, wherein the HSD17B13 gene is a human HSD17B13 gene. Embodiment 4. The isolated nucleic acid molecule comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 4. The nucleic acid molecule of any one of embodiments 1 to 3, comprising or consisting of 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, at least 11000, at least 12000, at least 13000, at least 14000, at least 15000, at least 16000, at least 17000, at least 18000 or at least 19000 nucleotides.

[0239] Embodiment 5. The nucleic acid molecule of any one of embodiments 1 to 4, wherein the isolated nucleic acid molecule comprises or consists of an HSD17B13 minigene in which one or more non-essential segments of the gene are deleted compared to the corresponding wild-type HSD17B13 gene.

[0240] Embodiment 6. The nucleic acid molecule of embodiment 5, wherein the deleted segment comprises one or more intron sequences. Embodiment 7. The nucleic acid molecule of embodiment 5 or 6, wherein the isolated nucleic acid molecule further comprises an intron corresponding to intron 6 of SEQ ID NO:2.

[0241] Embodiment 8. The nucleic acid molecule of embodiment 7, wherein the intron is intron 6 of SEQ ID NO:2. Embodiment 9. A nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides encoding all or a portion of an HSD17B13 protein, wherein the contiguous nucleic acid molecule comprises a segment that is 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 corresponding segment present in i) SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24 or SEQ ID NO:33 (transcript D), ii) SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:28 or SEQ ID NO:37 (transcript G), or iii) SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:29 or SEQ ID NO:38 (transcript H) that is not present in SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:21 or SEQ ID NO:30 (transcript A).

[0242] Embodiment 10. The nucleic acid molecule of embodiment 9, further comprising or consisting of a segment whose contiguous nucleotides are 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 corresponding segment present in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24 or SEQ ID NO:33 (transcript D) that is not present in SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:29 or SEQ ID NO:38 (transcript H), and further comprising or consisting of a segment whose contiguous nucleotides are 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 corresponding segment present in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24 or SEQ ID NO:33 (transcript D) that is not present in SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:28 or SEQ ID NO:37 (transcript G).

[0243] Embodiment 11. The nucleic acid molecule of embodiment 9, further comprising or consisting of a segment whose consecutive nucleotides are 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 corresponding segment present in SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:29, or SEQ ID NO:38 (transcript H) that is not present in SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:24, or SEQ ID NO:33 (transcript D).

[0244] Embodiment 12. The nucleic acid molecule of embodiment 9, wherein the contiguous nucleotides further comprise or consist of a segment that is 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 corresponding segment present in SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 28, or SEQ ID NO: 37 (transcript G) that is not present in SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, or SEQ ID NO: 33 (transcript D).

[0245] Embodiment 13. A nucleic acid molecule comprising at least 15 contiguous nucleotides encoding all or a part of an HSD17B13 protein, wherein the contiguous nucleotides comprise or consist of a segment that is 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 corresponding segment present in SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:25 or SEQ ID NO:34 (transcript E) that is not present in SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:21 or SEQ ID NO:30 (transcript A), and optionally, the contiguous nucleotides further comprise or consist of a segment that is 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 corresponding segment present in SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:25 or SEQ ID NO:34 (transcript E) that is not present in SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:29 or SEQ ID NO:38 (transcript H).

[0246] Embodiment 14. A nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides encoding all or a portion of an HSD17B13 protein, wherein the contiguous nucleotides comprise or consist of a segment that is 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 corresponding segment present in SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26 or SEQ ID NO:35 (transcript F) that is not present in SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:21 or SEQ ID NO:30 (transcript A).

[0247] Embodiment 15. A nucleic acid molecule comprising or consisting of at least 15 contiguous nucleotides encoding all or a portion of an HSD17B13 protein, wherein the contiguous nucleotides comprise or consist of a segment that is 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 corresponding segment present in SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:23 or SEQ ID NO:32 (transcript C) that is not present in SEQ ID NO:3, SEQ ID NO:12, SEQ ID NO:21 or SEQ ID NO:30 (transcript A).

[0248] Embodiment 16. The nucleic acid molecule of any one of embodiments 9 to 15, wherein the HSD17B13 protein is a human HSD17B13 protein. Embodiment 17. The nucleic acid molecule of any one of embodiments 9 to 16, wherein the isolated nucleic acid molecule comprises or consists of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000 or at least 2000 contiguous nucleotides encoding all or a portion of an HSD17B13 protein.

[0249] Embodiment 18. i) SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 23 or SEQ ID NO: 32 (transcript C), ii) SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24 or SEQ ID NO: 33 (transcript D), iii) SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25 or SEQ ID NO: 34 (transcript E), iv) SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26 or SEQ ID NO: 35 (transcript F), v) SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 28 or SEQ ID NO: 37 (transcript G), or vi) SEQ ID NO: 11, SEQ ID NO: 20, SEQ ID NO: 29 or SEQ ID NO: 38 (transcript H). a nucleic acid molecule encoding an HSD17B13 protein comprising or consisting of a sequence that is 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 sequence set forth in SEQ ID NO: 41 (isoform C), SEQ ID NO: 42 (isoform D), SEQ ID NO: 43 (isoform E), SEQ ID NO: 44 (isoform F), SEQ ID NO: 46 (isoform G) or SEQ ID NO: 47 (isoform H).

[0250] Embodiment 19. The nucleic acid molecule of any one of embodiments 9 to 18, wherein the consecutive nucleotides comprise or consist of the sequence of at least two different exons of the HSD17B13 gene, without intervening introns.

[0251] Embodiment 20. A polypeptide encoded by a nucleic acid molecule according to any one of embodiments 1 to 19. Embodiment 21. A nucleic acid molecule comprising or consisting of at least 15 consecutive nucleotides that hybridize to the HSD17B13 gene in a segment comprising, or within 1000 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 50 nucleotides, 45 nucleotides, 40 nucleotides, 35 nucleotides, 30 nucleotides, 25 nucleotides, 20 nucleotides, 15 nucleotides, 10 nucleotides, or 5 nucleotides of a position corresponding to position 12666 of SEQ ID NO: 2.

[0252] Embodiment 22. The nucleic acid molecule of embodiment 21, wherein the segment is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the corresponding sequence in SEQ ID NO:2 and has a thymine at a position corresponding to position 12666 of SEQ ID NO:2.

[0253] Embodiment 23. The nucleic acid molecule of embodiment 21 or 22, wherein the segment comprises or consists of at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000 consecutive nucleotides of SEQ ID NO:2.

[0254] Embodiment 24. A nucleic acid molecule according to any one of embodiments 21 to 23, wherein the segment comprises a ...

Claims

1. Use of an antisense molecule in the manufacture of a medicament for the treatment of a human subject who is not a carrier of the HSD17B13 rs72613567 variant and who is suffering from liver disease, wherein the antisense molecule hybridizes to a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) nucleic acid molecule and reduces expression of HSD17B13 in liver cells.

2. The use described in claim 1, wherein the antisense molecule hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A) or a sequence including part of exon 6 and part of exon 7 and reduces expression of HSD17B13 transcript A in hepatocytes in the human subject.

3. The use described in claim 2, wherein the antisense molecule comprises an antisense RNA that hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A).

4. The use described in claim 2, wherein the antisense molecule comprises an antisense RNA that hybridizes to a sequence including part of exon 6 and part of exon 7 of sequence number 12 (HSD17B13 transcript A).

5. The use described in claim 2, wherein the antisense molecule comprises an siRNA that hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A).

6. The use described in claim 2, wherein the antisense molecule comprises an siRNA that hybridizes to a sequence including part of exon 6 and part of exon 7 of sequence number 12 (HSD17B13 transcript A).

7. The use described in claim 2, wherein the antisense molecule comprises an shRNA that hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A).

8. The use described in claim 2, wherein the antisense molecule comprises an shRNA that hybridizes to a sequence including part of exon 6 and part of exon 7 of sequence number 12 (HSD17B13 transcript A).

9. The use described in claim 1, wherein the liver disease is a chronic liver disease.

10. The use of claim 1, wherein the liver disease is fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple fatty liver, steatohepatitis, fibrosis or non-alcoholic steatohepatitis (NASH).

11. The use described in claim 10, wherein the liver disease is NAFLD.

12. The use described in claim 10, wherein the liver disease is alcoholic fatty liver disease.

13. The use described in claim 10, wherein the liver disease is cirrhosis.

14. The use described in claim 10, wherein the liver disease is hepatocellular carcinoma.

15. Use of an antisense molecule in the manufacture of a medicament for preventing liver disease in a human subject who is not a carrier of the HSD17B13 rs72613567 variant, wherein the antisense molecule hybridizes to a hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) nucleic acid molecule and reduces expression of HSD17B13 in liver cells.

16. The use of claim 15, wherein the antisense molecule hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A) or a sequence including part of exon 6 and part of exon 7 and reduces expression of HSD17B13 transcript A in hepatocytes in the human subject.

17. The use described in claim 16, wherein the antisense molecule comprises an antisense RNA that hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A).

18. The use described in claim 16, wherein the antisense molecule comprises an antisense RNA that hybridizes to a sequence including part of exon 6 and part of exon 7 of sequence number 12 (HSD17B13 transcript A).

19. The use described in claim 16, wherein the antisense molecule comprises an siRNA that hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A).

20. The use described in claim 16, wherein the antisense molecule comprises an siRNA that hybridizes to a sequence including part of exon 6 and part of exon 7 of sequence number 12 (HSD17B13 transcript A).

21. The use described in claim 16, wherein the antisense molecule comprises an shRNA that hybridizes to a sequence within exon 7 of SEQ ID NO: 12 (HSD17B13 transcript A).

22. The use described in claim 16, wherein the antisense molecule comprises an shRNA that hybridizes to a sequence including part of exon 6 and part of exon 7 of sequence number 12 (HSD17B13 transcript A).

23. The use of claim 15, wherein the liver disease includes chronic liver disease.

24. The use of claim 15, wherein the liver disease comprises fatty liver disease, non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, cirrhosis, viral hepatitis, hepatocellular carcinoma, simple fatty liver, steatohepatitis, fibrosis or non-alcoholic steatohepatitis (NASH).

25. The use described in claim 24, wherein the liver disease includes NAFLD.

26. The use described in claim 24, wherein the liver disease includes alcoholic fatty liver disease.

27. The use of claim 24, wherein the liver disease includes cirrhosis.

28. The use of claim 24, wherein the liver disease includes hepatocellular carcinoma.

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