Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-14
2003-04-08
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06544743
ABSTRACT:
BACKGROUND OF THE INVENTION
Peroxisome proliferator-activated receptors (PPARs) are members of a large family of ligand-inducible transcription factors that includes receptor for retinoids and vitamin D as well as thyroid and steroid hormones (1). PPARs regulate the expression of target genes by binding to DNA sequence elements, termed PPAR response elements (PPRE) (2,3). PPREs have been identified in the regulatory regions of a variety of genes involved in lipid and energy balance. Three different PPAR genes have been identified (&agr;, &dgr; and &ggr;) (2,3). PPAR&ggr;, expressed mainly in adipose tissue, regulates adipocyte differentiation (4,5). PPAR&agr;, the first to be identified, is expressed mainly in tissues that have a high level of fatty acid (FA) catabolism such as the liver, kidney, heart and muscle (6,7). Numerous studies have demonstrated that several genes encoding enzymes involved in &bgr; and &ohgr;-oxidation are under the control of PPAR&agr; (2,3). PPAR&agr; is activated by medium and very long chain FA and polyunsaturated FA, such as eicosapentaenoic acid (8). In addition to its role in FA oxidation, PPAR&agr; appears to play an important role in the control of extracellular lipid metabolism. In fact, a variety of hypolipidemic fibrates are synthetic ligands for PPAR&agr; (9).
FAs are essential biological components and are used as metabolic fuels, covalent regulators of signaling molecules, and essential components of cellular membranes. Thus, FA must be kept within narrow physiological limits. Altered levels of FA are linked to a variety of metabolic diseases including atherosclerosis, hyperlipidemia, obesity, insulin resistance and type 2 diabetes (10, 11).
SUMMARY OF THE INVENTION
Peroxisome proliferator-activated receptor alpha (PPAR&agr;) is a member of the steroid hormone receptor super family that is involved in the control of cellular lipid utilization. As described herein, the genomic structure of the human PPAR&agr; was determined, and intronic primers were designed to sequence the complete coding region and the exon-intron boundaries of the human PPAR&agr; gene among 12 patients with type 2 diabetes and 2 controls. Sequence analyses revealed the presence of a L162V missense mutation in exon 5; that is, there is a C to G transversion in the first position of codon 162 leading to a substitution of a valine for the leucine at that position. Mutation L162V is contained within the DNA binding domain of the human PPAR&agr; gene, and leucine 162 is highly conserved among humans, mice, rats and guinea pigs. Among diabetics and controls, a trend was observed toward higher plasma LDL-cholesterol and apolipoprotein B levels among carriers of the rare V162 allele. To verify these findings, an independent cohort of 193 non-diabetic subjects recruited in the greater Quebec City area was screened. Comparison of the lipoprotein-lipid profile between L162 homozygotes and carriers of the rare V 162 allele showed significant differences in plasma LDL-cholesterol, total and LDL-apolipoprotein B concentrations, carriers of the V 162 allele having the highest levels. These results suggest that the rare V162 allele may make the subjects more prone to develop a hyperapobetalipoproteinemia.
Thus, the invention relates to the SNPs identified as described herein, both singly and in combination, as well as to the use of these SNPs, and others in PPAR (e.g., PPAR&agr;) genes, particularly those nearby in linkage disequilibrium with these SNPs, for diagnosis, prediction of clinical course and treatment response for disorders of lipid metabolism, development of new treatments for disorders of lipid metabolism based upon comparison of the variant and normal versions of the gene or gene product, and development of cell-culture based and animal models for research and treatment of disorders of lipid metabolism. The invention also relates to methods for diagnosing and treating disorders of lipid metabolism, especially high LDL cholesterol levels and hyperapobetalipoproteinemias, and to methods for identifying compounds for use in the diagnosis and treatment of said disorders. The invention further relates to novel compounds and pharmaceutical compositions for use in the diagnosis and treatment of disorders of lipid metabolism.
The invention relates to isolated nucleic acid molecules comprising all or a portion of the variant allele of PPAR&agr; (e.g., wherein reference or wildtype PPAR&agr; is exemplified by SEQ ID NO: 1). Preferred portions are at least 10 contiguous nucleotides and comprise the polymorphic site, e.g., a portion of SEQ ID NO: 1 which is at least 10 contiguous nucleotides and comprises the “G” at the first position of codon 162 (nucleotide 696) of the PPAR&agr; gene, or a portion of SEQ ID NO: 1 which is at least 10 contiguous nucleotides and comprises the “A” at the last position of codon 253 (nucleotide 971) of the PPAR&agr; gene. The invention further relates to isolated gene products, e.g., polypeptides or proteins, which are encoded by a nucleic acid molecule comprising all or a portion of the variant allele of PPAR&agr;.
The invention further relates to isolated proteins or polypeptides comprising all or a portion of the variant amino acid sequence of PPAR&agr; (e.g., wherein reference or wildtype PPAR&agr; is exemplified by SEQ ID NO: 2), and to isolated proteins or polypeptides comprising all or a portion of the variant amino acid sequence of PPAR&agr;. Preferred polypeptides are at least 10 contiguous amino acids and comprise the polymorphic amino acid, e.g., a portion of SEQ ID NO: 2 which is at least 10 contiguous amino acids and comprises the valine at residue 162. The invention further relates to isolated nucleic acid molecules encoding such proteins and polypeptides, as well as to antibodies which bind, e.g., specifically, to such proteins and polypeptides.
In one embodiment, the invention relates to a method for predicting the likelihood that an individual will have a disorder of lipid metabolism (or diagnosing or aiding in the diagnosis of a disorder of lipid metabolism), e.g., high LDL cholesterol levels, atherosclerosis, coronary heart disease and/or hyperapobetalipoproteinemias, comprising the steps of obtaining a DNA sample from an individual to be assessed and determining the nucleotide present at the first position of codon 162 (nucleotide 696) of the PPAR&agr; gene. The presence of a “C” (the reference nucleotide) at this position indicates that the individual has a lower likelihood of having a disorder of lipid metabolism than an individual having a “G” at that position, or a greater likelihood of having less severe symptomology. In a preferred embodiment, the disorder is high LDL cholesterol levels, atherosclerosis and/or coronary heart disease. In another embodiment, the disorder is a hyperapobetalipoproteinemia. In a particular embodiment, the individual is an individual at risk for development of a disorder of lipid metabolism.
In another embodiment, the invention relates to a method for predicting the likelihood that an individual will have a disorder of lipid metabolism (or diagnosing or aiding in the diagnosis of a disorder of lipid metabolism), e.g., high LDL cholesterol levels, atherosclerosis, coronary heart disease and/or hyperapobetalipoproteinemias, comprising the steps of obtaining a DNA sample from an individual to be assessed and determining the nucleotide present at the first position of codon 162 (nucleotide 696) of the PPAR&agr; gene. The presence of a “G” (the variant nucleotide) at this position indicates that the individual has a greater likelihood of having a disorder of lipid metabolism than an individual having a “C” at that position, or a greater likelihood of having more severe symptomology. In a preferred embodiment, the disorder is high LDL cholesterol levels, atherosclerosis and/or coronary heart disease. In another embodiment, the disorder is a hyperapobetalipoproteinemia. In a particular embodiment, the individual is an individual at risk for development of a disorder of lipid metabolism.
In another embod
Brewer Carl
Gaudet Daniel
Hudson Thomas J.
Morgan Kenneth
Vohl Marie-Claude
Hamilton Brook Smith & Reynolds P.C.
McGill University
Myers Carla J.
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