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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/172—Haplotypes

Definitions

• the field of the invention is sequence variations in the human gene PNPLA3 as risk factors for hepatic steatosis and hepatic injury.
• Nonalcoholic fatty liver disease is a burgeoning health problem of unknown etiology that varies in prevalence among ethnic groups.
• NAFLD Nonalcoholic fatty liver disease
• variation in PNPLA3 contributes to ethnic and inter-individual differences in hepatic fat content and susceptibility to NAFLD.
• the invention provides methods and compositions for identifying a genetic variant in a person determined to have or be predisposed to having a liver disease, the method comprising the step of determining whether the person has PNPLA3-I148M.
• the invention provides methods and compositions for identifying a genetic variant in a person, the method comprising the steps of (a) determining whether the person has PNPLA3-I148M; and (b)(i) providing the person with information about risk of developing a liver disease, or (ii) providing the person with a recommendation for an additional diagnostic test or monitoring to detect an indication of the liver disease, or (iii) prescribing to the person a treatment for the liver disease.
• the liver disease is fatty liver, an increase in or supra-normal hepatic fat, hepatic steatosis, steatohepatitis, nonalcoholic fatty liver disease, or associated inflammation.
• the methods further comprise the step of determining whether the person has PNPLA3-S453I.
• the determining step comprises detecting the PNPLA3-I148M and/or PNPLA3-S453I using a method selected from the group consisting of: mass spectroscopy, oligonucleotide microarray analysis, allele-specific hybridization, allele-specific PCR, and sequencing.
• the determining step comprises detecting a marker of PNPLA3-I148M that is SNP rs738408, or a surrogate SNP in linkage disequilibrium with the PNPLA3-I148M and having a r 2 value greater than 0.5.
• rs738408 1
• associated SNPs having a r 2 value greater than 0.5 In Table 1 below, the rs number is shown, followed by the correlation with the 1148M allele (r 2 value). These data are from Caucasians in the HapMap.
• the determining step comprises detecting a plurality of the SNP and surrogate SNP markers.
• the methods further comprise an antecedent step of determining that the person has or is predisposed to having a subject liver disease.
• the subject liver diseases are amenable to convention clinical diagnosis; for example, fatty liver or hepatic steatosis may be determined inter alia using computer-aided tomography (CAT) scan or NMR, such as proton magnetic resonance spectroscopy, and is generally clinically defined as hepatic triglyceride greater than 5.5%.
• Indicators of predisposition to fatty liver include obesity, diabetes, insulin resistance, and alcohol ingestion.
• the methods may further comprise the step of prescribing to the person a treatment for the liver disease or treating the person with a therapy for the liver disease, such an anti-obesity drug such as Orlistat, Sibutramine, Byetta, Symlin, Rimonabant, or an anti-diabetic drugs such as a thiazolidinedione (e.g. rosiglitazone), or metformin or glimepiride, or anti-inflammatory drugs.
• a treatment for the liver disease or treating the person with a therapy for the liver disease
• a therapy for the liver disease such an anti-obesity drug such as Orlistat, Sibutramine, Byetta, Symlin, Rimonabant, or an anti-diabetic drugs such as a thiazolidinedione (e.g. rosiglitazone), or metformin or glimepiride, or anti-inflammatory drugs.
• the invention provides methods and compositions for identifying a genetic variant in a person determined to have or be predisposed to having a subnormal hepatic fat or triglyceride content, or a subnormal susceptibility to hepatic steatosis or nonalcoholic fatty liver disease, the method comprising the step of determining whether the person has PNPLA3-S453I.
• the invention provides methods and compositions for identifying a genetic variant in a person, the method comprising the steps of: (a) determining whether the person has PNPLA3-S453I; and (b) providing the person with information about predisposition to have a subnormal hepatic fat or triglyceride content or a subnormal susceptibility to hepatic steatosis or nonalcoholic fatty liver disease, or providing the person with a recommendation for an additional diagnostic test or monitoring to detect an indication of liver disease, or prescribing to the person an alternative treatment for liver disease.
• the determining step comprises detecting the variant using a method selected from the group consisting of: mass spectroscopy, oligonucleotide microarray analysis, allele-specific hybridization, allele-specific PCR, and sequencing.
• the determining step comprises detecting a marker of PNPLA3-S453I that is SNP rs6006460, or a surrogate SNP in linkage disequilibrium with the PNPLA3-S453I and having a r 2 value greater than 0.5.
• the determining step comprises detecting a plurality of the SNP and surrogate SNP markers.
• the invention provides reagents and kits for practicing the disclosed methods.
• adipose tissue serves as a reservoir to limit the deposition of triglyceride (TG) in the liver and other metabolically active tissues 1 .
• TG triglyceride
• the effectiveness of this buffer in protecting against the accumulation of fat in the liver varies widely among individuals: hepatic fat content ranges from less than 1% to more than 50% of liver weight in the general population 2 .
• the accumulation of excess TG in the liver a condition known as hepatic steatosis (or fatty liver), is associated with adverse metabolic consequences, including insulin resistance and dyslipidemia 3,4 .
• Nonalcoholic fatty liver disease is the most common form of liver disease in Western countries 6 . Approximately 10% of liver transplants performed in the United States are for cirrhosis related to NAFLD 4 .
• Factors promoting deposition of fat in the liver include obesity, diabetes, insulin resistance, and alcohol ingestion 3,6 . Hispanics are particularly susceptible to develop fatty liver and also have a higher prevalence of steatohepatitis and cirrhosis, whereas African-Americans tend to be resistant to the accumulation of liver fat and are less prone to develop liver failure 2,7-9 .
• the variant is a cytosine to guanine substitution that changes codon 148 from isoleucine to methionine; this residue is highly conserved in vertebrates.
• PNPLA3 encodes a 481 amino acid protein of unknown function that belongs to the patatin-like phospholipase family 14 .
• ALT alanine aminotransferase
• Increased hepatic fat content is associated with insulin resistance and dyslipidemia (increased plasma levels of TG and lower levels of high density lipoprotein-cholesterol), but the causal nature of these relationships remains poorly defined 3 .
• No association was found between the PNPLA3-I148M allele and body mass index (BMI) or indices of insulin sensitivity, including fasting glucose and insulin or homeostatic model assessment of insulin resistance (HOMA-IR) in the Dallas Heart Study. No associations were observed between PNPLA3 genotype and plasma levels of TG, total cholesterol, HDL-cholesterol or LDL-cholesterol.
• the identification of a second allele of PNPLA3 that was independently associated with liver fat content further supports a role for PNPLA3 in determining liver fat levels, and indicates the presence of both loss-of-function and gain-of-function alleles at this locus.
• PNPLA3 is under metabolic control in adipose tissue and the liver, with levels being low in the fasted state and increases dramatically with carbohydrate feeding 20,21 .
• PNPLA3 structurally resembles calcium-independent phospholipase A 2 but the recombinant protein has low phospholipase activity when expressed in insect (Sf9) cells 22 .
• PNPLA3 has more robust activity against TG in vitro and can also transfer fatty acids to and from mono- and diacylglycerol 22 .
• the Dallas Heart Study is a population-based probability sample of Dallas County.
• the sampling frame and the study design are described in detail in Victor et al. 10 African-Americans were over-sampled (52% African American, self-identified as ‘black’, 29% European American, self-identified as ‘white’, 17% Hispanic self identified as “Hispanic” and 2% other ethnicities).
• the institutional review board of University of Texas Southwestern Medical Center approved the study. Alcohol consumption was determined according to answers to previously validated questions 2 .
• Blood pressure, height, weight and BMI and calculated variables were measured as described 10 Fasting blood samples were obtained from 3,551 subjects (ages 30-65) and 2,971 of these individuals completed a clinic visit; hepatic TG content was measured using 1 H-MRS in 2,240 African-Americans, European-Americans and Hispanics 7,12 .
• ARIC Atherosclerosis Risk in Communities Study
• PNPLA3 Resequencing PNPLA3.
• the exons and flanking introns of PNPLA3 were sequenced as described previously 28 in the African-American, European-American and Hispanic men and women in the Dallas Heart Study with the highest and lowest hepatic TG content. All sequence variants identified were verified by manual inspection of the chromatograms and missense changes were confirmed by an independent resequencing reaction.
• Genotyping assays Fluorogenic 5′-nucleotidase assays were developed for PNPLA3-I148M and for the sequence variants identified in both the high and low hepatic TG groups in the resequencing experiments. Sequence variations in PNPLA3 were assayed using the TaqMan assay system (Applied Biosystems) on a 7900HT Fast Real-Time PCR instrument. Probes and reagents were purchased from Applied Biosystems.

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• 2009
  • 2009-09-03 US US12/553,330 patent/US20100056384A1/en not_active Abandoned
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• 2012
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