Repeat polymorphism in the frataxin gene and uses therefore

Details Repeat polymorphism in the frataxin gene and uses therefore Repeat polymorphism in the frataxin gene and uses therefore

1999-04-15

2001-11-27

Schwartzman, Robert A. (Department: 1632)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S024310, C536S024330, C435S091200, C435S091500

Reexamination Certificate

active

06322978

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a frataxin intermediate GAA repeat length polymorphism and uses therefore, e.g., genetic screening of subjects at risk for non insulin diabetes mellitus.
BACKGROUND OF THE INVENTION
Non insulin diabetes mellitus (NIDDM) is a clinically heterogeneous disorder (Kahn, C. R. (1994)
Diabetes
43:1066-1084) characterized by insulin resistance in peripheral tissues combined with impaired insulin secretion of the pancreatic &bgr;-cell (DeFronzo, R. A. et al. (1992)
Diabetes Care
15:318-368). Investigations of candidate genes for NIDDM have led to the identification of polymorphisms of several genes encoding proteins of insulin action and insulin secretion, but none show a high frequency of association with the disease (Kahn, C. R. et al. (1996)
Annu. Rev. Med.
47:509-531; McCarthy, M. I. et al. (1994)
Diabetologia
37:959-968).
Friedreich's ataxia (FRDA) is the most common hereditary ataxia (Campuzano, V. et al. (1996)
Science
271:1423-1427), with an estimated prevalence of 1 in 50,000 people (Epplen, C. et al. (1997)
Hum. Genet.
99:834-836). FRDA is a degenerative disease characterized by progressive ataxia, lack of tendon reflexes, loss of position sense, dysarthria, and hypertrophic cardiomyopathy. FRDA is inherited as an autosomal recessive disease. FRDA appears to belong to the family of triplet repeat disorders, including myotonic dystrophy, fragile X syndrome, and Huntington's disease (Wells, R. D. (1996)
J. Biol. Chem.
271:2875-2878). The FRDA has been mapped to chromosome 9q13-q21 (Hanauer, A. et al. (1990)
Am. J. Hum. Genet.
46:133-137) and attributed to a GAA triplet repeat expansion (McCarthy, M. I. et al. (1994)
Diabetologia
37:959-968) in the frataxin gene, also known as X25 or STM7. Frataxin encodes a protein, which is targeted to the mitochondria and has been suggested to function as an endogenous antioxidant (Rotig, A. et al. (1997)
Nat. Genet.
17:215-217).
SUMMARY OF THE INVENTION
In general, the invention features, a method for determining whether a subject is at risk for a disorder characterized by an intermediate GAA repeat length polymorphism in the frataxin gene. The method includes: obtaining a biological sample from the subject; and detecting the presence or absence of an intermediate GAA repeat length polymorphism in frataxin nucleic acid in the biological sample e.g. in genomic DNA, thereby determining whether the subject is at risk for a disorder characterized by an intermediate GAA repeat length polymorphism in the frataxin gene.
In preferred embodiments, the disorder characterized by an intermediate GAA repeat length polymorphism in the frataxin gene is diabetes, e.g., non-insulin dependent diabetes mellitus or a disorder characterized by insufficient levels of an anti-oxidant.
In preferred embodiments, the method includes isolating a nucleic acid, e.g., DNA, total RNA, or mRNA, from the biological sample. For example, mRNA can be isolated using the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979)
Biochemistry
18: 5294-5299.
In preferred embodiments, the presence or absence of an intermediate GAA repeat length polymorphism in the frataxin gene is detected by the use of a polymerase chain reaction, e.g., by the use of primers which can hybridize to a frataxin gene containing an intermediate length GAA repeat and e.g., produce a product characteristic of the presences of the intermediate GAA repeat.
In preferred embodiments, the presence or absence of an intermediate GAA repeat length polymorphism in the frataxin gene is detected by an alteration in a restriction enzyme cleavage pattern. For example, DNA can be isolated from a biological sample, cleaved with one or more restriction enzymes, and then compared to a control for the pattern of migration on a gel, e.g., an agarose gel.
In preferred embodiments, the presence or absence of an intermediate GAA repeat length polymorphism in the frataxin gene is detected by nucleic acid sequencing, e.g., DNA sequencing. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977)
PNAS
74:560) or Sanger ((1977)
PNAS
74:5463). A variety of automated sequencing procedures can also be used, including sequencing by mass spectrometry (described in, for example, PCT International Publication No. WO 94/16101; Cohen et al. (1996)
Adv. Chromatogr.
36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol
38:147-159).
In preferred embodiments, the biological sample is a biological fluid, e.g., blood, plasma, cerebrospinal fluid, or urine; a tissue sample, e.g., a skin biopsy sample; or a cellular sample, e.g., a blood cell sample or a skin cell sample.
In preferred embodiments, the subject can be a human, an experimental animal, e.g., a rat or a mouse, a domestic animal, e.g., a dog, cow, sheep, pig or horse, or a non-human primate, e.g., a monkey.
In preferred embodiments the method further includes: the step of identifying an individual as at risk for NIDDM or an antioxidant-related disorder; the step of administering a second diagnostic test for NIDDM or an antioxidant-related disorder; testing the subject for insulin resistance; testing the subject for glucose tolerance.
In another aspect, the invention features, a method for detecting the presence of a frataxin intermediate GAA repeat length polymorphism in a biological sample. The method includes: contacting a biological sample with an agent capable of detecting a frataxin intermediate GAA repeat length polymorphism such that the presence of a frataxin intermediate GAA repeat length polymorphism is detected in the biological sample.
In preferred embodiments: the agent is a labeled or labelable nucleic acid probe capable of hybridizing to frataxin mRNA or DNA; the agent is a nucleic acid probe capable of amplifying a frataxin gene containing an intermediate length GAA repeat.
In another aspect, the invention features, a kit for detecting the presence of a frataxin intermediate GAA repeat length polymorphism in a biological sample including an agent capable of detecting a frataxin intermediate GAA repeat length polymorphism in a biological sample.
In preferred embodiments: the kit further includes means for determining the amount of a frataxin intermediate GAA repeat length polymorphism in the sample and means for comparing the amount of a frataxin intermediate GAA repeat length polymorphism in the sample with a standard; the agent is a nucleic acid probe capable of amplifying a frataxin gene containing an intermediate length GAA repeat.
In preferred embodiments the method further includes: the step of identifying an individual as at risk for NIDDM or an antioxidant-related disorder; the step of administering a second diagnostic test for NIDDM or an antioxidant-related disorder; testing the subject for insulin resistance; testing the subject for glucose tolerance.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for NIDDM or an antioxidant-related disorder. The method includes examining the subject for the expression of the frataxin gene product, non-wild type expression or mis-expression, particularly lowered expression, and more particularly lowered but not abolished expression, being indicative of risk. Expression can be detected at the protein or mRNA level.
In preferred embodiments the method further includes: the step of identifying an individual as at risk for NIDDM or an antioxidant-related disorder; the step of administering a second diagnostic test for NIDDM or an antioxidant-related disorder; testing the subject for insulin resistance; testing the subject for glucose tolerance.
In another aspect, the invention provides a substantially pure nucleic acid having, or comprising, an intermediate GAA repeat.
In preferred embodiments, the nucleic acid further includes: the first intron of frataxin; a sequence, e.g., a genomic sequence, which encodes frataxin; preferably including the first intron of frataxin; the first intron of frataxin fused (with or wi

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