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Isolated nucleic acids and polypeptides associated with glucose homeostasis disorders and method of detecting the same

Isolated nucleic acids and polypeptides associated with glucose homeostasis disorders and method of detecting the same description/claims

This application is based on and claims priority to U.S. Provisional Application Ser. No. 60/215,477, filed Jun. 29, 2000, herein incorporated by reference in its entirety.

The present invention relates generally to nucleic acid sequences coding for sugar transporters useful for screening against glucose homeostasis disorders, and particularly to nucleic acid sequences coding for the glucose transporter which map to the region between D20S119 and D20S178 on human chromosome 20q13.

The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the text.

Diabetes mellitus is a condition in which the glucose homeostasis of a subject becomes unbalanced and leads to a hyperglycemic systemic condition. There are two forms of the diabetic condition, Type I and Type II. Type I diabetes usually occurs in individuals under approximately 20 years of age, is insulin-dependent, is commonly accompanied by ketoacidosis and represents about 10% of the diabetic population. Type II diabetes affects approximately 5 percent of the adult American population and represents about 90% of the diabetic population. Type II diabetes is commonly associated with obesity, usually occurs in individuals over approximately 40 years of age and is non-insulin dependent. A subset of type II diabetes can occur in younger individuals and is referred to as maturity onset diabetes of the young (MODY).

Interestingly, persons suffering from Type II diabetes can exhibit normal or even elevated levels of insulin, which helps the body maintain glucose homeostasis. This suggests that in Type II diabetes there might be a decreased sensitivity of the body to the effects of insulin, although defective insulin secretion can also be involved. Another critical factor in systemic maintenance of glucose homeostasis is the uptake of glucose by glucose transporter proteins. Furthermore, other factors include excessive hepatic glucose production and increased lipolysis in adipose tissue. See, e.g., Edelman, (1998) Adv. Internal Med. 43: 449-500; Vaag, (1999) Dan. Med. Bull. 46 (3): 197-234.

Type II diabetes is typically slow to develop, is hereditary and is associated with obese individuals. Ongoing research has demonstrated that there is both a genetic component and an environmental component that leads to the Type II diabetic condition. The environmental component can lead to an acquired resistance to the action of insulin. The genetic component manifests itself as a condition rendering an individual predisposed to insulin resistance and more susceptible to the chronic onset of the diabetic condition. The genetic component can also involve glucose uptake by glucose transport proteins. Studies of the Pima Indian population, which has an unusually high incidence of Type II diabetes, and other populations, have indicated that alterations in glucose metabolism can be detected in subjects monitored before the onset of the condition. The genetic component presents a complex pattern of inheritance that is not fully understood.

Studies of the genetic component of the condition have indicated linkage of Type II diabetes susceptibility on many human chromosomes, including 2, 11, 12 and 20. A locus on chromosome 2 appears to be a major factor in development of Diabetes Mellitus in Mexican Americans (Hanis et al., (1996) Nat. Genet. 13: 161-66), a locus on chromosome 12 has been found in a Finnish populations (Mahtani et al., (1996) Nat. Genet. 14: 90-94) and a locus on chromosome 11q has been identified in Pima Indians (Hanson et al., (1998) Diabetes 46: 494-501). Studies by Zouali et al. suggest the location of a susceptibility locus on chromosome 20q in the PCK1 region. Zouali et al., (1997) Hum. Mol. Genet. 6:1401-08. At least three other studies present evidence for linkage of Type II diabetes on chromosome 20 in sibships. Ghosh et al., (1999) Proc. Natl. Acad. Sci. USA 96: 2198-2203; Bowden et al., (1997) Diabetes 46: 882-86; Ji et al., (1997) Diabetes 46: 876-81.

As noted, a hallmark of the Type II diabetic condition is a hyperglycemic imbalance in body glucose homeostasis. This condition can arise from a defect in the mechanism by which glucose is processed by the human body and can occur at various control points in the mechanism. A defect can occur, for example, during glucose uptake by the brain, glucose storage in the liver or insulin-dependent uptake in muscles and adipocytes in the human body.

Central to the uptake of glucose and the maintenance of glucose homeostasis are the glucose transporter proteins. These proteins control glucose absorption by the above-mentioned tissues. In mammalian cells, glucose transport is catalyzed by a number of membrane proteins, including the GLUT family of proteins, GLUT1-5, GLUT8, GLUTX1 and GLUT9. Glucose transport proteins are found in a wide variety of species, and share a common structural motif: glucose transport proteins are characterized by the presence of 12 connected transmembrane helical segments. See, e.g., Ibberson et al., (2000) J. Biol. Chem. 275: 4607-12; Doege et al., (2000) J. Biol. Chem. 275: 16275-80. An extracellular loop containing a glycosylation site is also present.

Although the precise primary defect in the Type II diabetic condition is presently unsettled, work in the field suggests that there is a strong causal link between the condition and glucose mobilization and metabolism. Zierler, (1999) Am. J. Physiol. 276 (3, pt. 1): E409-26; Shepard and Kahn, (1999) New Engl. J. Med. 341 (4): 248-57. Glucose transporter proteins are, therefore, likely candidates for analysis when attempting to explain the link between the Type II diabetic condition, as well as other glucose homeostatis imbalances, and their genetic components. Indeed, a mutation in GLUT4 has been found in a patient with Type II diabetes. Kusari, et al., (1991) JCI 88: 1323-30.

Clearly, it would be of tremendous value to researchers and clinicians to have a specific polynucleotide sequence coding for a glucose transport protein which is identified as being associated with human Type II diabetes. Such a result would permit diagnostic and therapeutic treatment of the condition, which if left untreated can lead to circulatory deficiencies and blindness. Traditional genetic approaches have proven to be inadequate to accomplish this goal. Linkage disequilibrium analysis, for example, which has historically proven helpful in studying complex disease genes, has not yet yielded results useful for Type II diabetes-related therapeutic treatments.

What is needed, therefore, is the identification of a polynucleotide sequence coding for a glucose transporter that is associated with human Type II diabetes and other glucose homeostasis disorders, as well as a method for accurately diagnosing a subject's susceptibility to, and the early detection of such conditions. Gene and drug therapy to relieve this condition, after the condition has manifested, is also needed. The present invention solves this problem by providing a polynucleotide and a polypeptide useful for diagnosing the susceptibility of a subject to human Type II diabetes and other glucose homeostasis disorders, a method for diagnosing the susceptibility of a subject to human Type II diabetes and other glucose homeostasis disorders using the polynucleotide and polypeptide and a method of treating the condition, all of which take into account the polymorphic nature of the polynucleotide sequence.

An isolated and substantially pure nucleic acid sequence located between D20S119 and D20S178 on human chromosome 20q13 is disclosed. The nucleic acid sequence comprises a nucleic acid sequence coding for a novel glucose transporting protein and having the sequence shown in SEQ ID NO: 1, or a nucleic acid sequence having at least 70% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. Additionally, a nucleic acid sequence which hybridizes to and is at least 70% complementary to the glucose transporter-encoding nucleic acid sequence is disclosed.

Also disclosed is a method of facilitating a diagnosis of susceptibility to a glucose homeostasis disorder. The method comprises obtaining a biological sample from a subject; isolating a target nucleic acid sequence located between D20S119 and D20S178 on human chromosome 20q13, the target nucleic acid sequence encoding a glucose transporter polypeptide; sequencing the target nucleic acid sequence; and determining a sequence variation between a wild type nucleic acid sequence and the isolated target nucleic acid sequence, the presence of variations between the wild type nucleic acid sequence and the isolated target nucleic acid sequence indicating susceptibility in the subject to a disorder affecting glucose homeostasis.

A method of screening a biological sample for the presence of the novel glucose transporter polypeptide is also disclosed. The presence of polypeptide in the sample is detected by evaluating the formation and presence of antibody-polypeptide conjugates.

In another aspect, the present invention provides a method of screening a biological sample for the presence of antibodies immunoreactive with a novel glucose transporter polypeptide. In accordance with such a method, a biological sample is exposed to a glucose transporter polypeptide under biological conditions and for a period of time sufficient for antibody-polypeptide conjugate formation and the formed conjugates are detected.

A method of a facilitating a diagnosis of a disorder affecting glucose homeostasis is also disclosed. In one embodiment, the method comprises: (a) obtaining a biological sample from a subject; and (b) determining an amount of a glucose transporter polypeptide present in the biological sample, wherein the presence of a reduced amount of the glucose transporter polypeptide as compared to a standard facilitates a diagnosis of a disorder affecting glucose homeostasis. Optionally, the amount of glucose transporting protein in the biological sample can be determined by Western blot analysis.

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