Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-08-18
2001-08-28
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C536S023100, C536S024310, C536S024330
Reexamination Certificate
active
06280938
ABSTRACT:
BACKGROUND OF THE INVENTION
Trinucleotide repeat expansions have been shown to be the mutational mechanism responsible for a growing number of diseases, including Fragile X mental retardation, spinobulbar muscular atrophy, myotonic dystrophy (DM), Huntington disease (HD), spinocerebellar ataxia (SCA) types 1, 2, 3 and 6, dentatorubral pallidoluysian atrophy and Friedreich's ataxia. A hallmark for most of these diseases is the presence of anticipation, or a decrease in the age of onset and increase in disease severity in consecutive generations due to the tendency for the unstable trinucleotide repeat tract to lengthen when passed from one generation to the next (Warren, S. T.
Science,
271, 1374-1375 (1996)).
In 1993, Schalling et al. (
Nature Genetics,
4, 135-139 (1993)) developed the repeat expansion detection (RED) assay. RED is an elegant technique that detects potentially pathological trinucleotide repeat expansions without prior knowledge of chromosomal location or flanking DNA sequence. Human genomic DNA is used as a template for a two-step ligation cycling process that generates sequence specific [(CAG)
n
, (CGG)
n
, etc.] oligonucleotide multimers when expanded trinucleotide sequences are present in the genome. The assay was originally developed to detect very large trinucleotide repeat expansions present in genomic DNA from patients with Myotonic Dystrophy (DM) and Fragile X syndrome (up to 2,000 repeats). Since that time, Lindblad et al. have modified the procedure to detect smaller trinucleotide repeats in the size range (40-100 CAG repeats) pathologic for SCA1, SCA3, HD, and SBMA (Lindblad, K., et al., Nature Genetics 7, 124 (1994), Lindblad, K. et al.,
Genome Research,
6, 965-971 (1996)).
This modified assay has been used to establish correlations that suggest the involvement of CAG expansions in diseases such as SCA7 (Lindblad, K. et al.,
Genome Research,
6, 965-971 (1996)), bipolar affective disorder (Oruc, L. et al.,
Am J Hum Genet.,
60, 732-735 (1997)) and schizophrenia (Maraganore, D. M., et al.,
Neurology,
47, (1996)).
The spinocerebellar ataxias (SCAs) are progressive degenerative neurological diseases of the nervous system characterized by a progressive degeneration of neurons of the cerebellar cortex. Degeneration is also seen in the deep cerebellar nuclei, brain stem, and spinal cord. Clinically, affected individuals suffer from severe ataxia and dysarthria, as well as from variable degrees of motor disturbance and neuropathy. The disease usually results in complete disability and eventually in death 10 to 30 years after onset of symptoms. The genes for SCA types 1, 2, 3 and 6 have been identified. All contain CAG DNA repeats that cause the disease when the repeat region is expanded. Little is known how CAG repeat expansion and elongation of polyglutamine tracts relate to neurodegeneration. The identification of the SCA7 gene would provide an opportunity to study this phenomenon in a new protein system.
The significance of identifying ataxia genes provides an improved method for diagnosis of individuals with the disease and increases the possibility of prenatal/presymptomatic diagnosis or better classification of ataxias. Most of the genes associated with repeat expansions in the coding region including the other SCA genes now identified, show no homology to known genes.
SUMMARY OF THE INVENTION
The present invention relates to methods for identifying individuals at risk and individuals not at risk for developing spinocerebellar ataxia type 7. These methods include the step of analyzing the CAG repeat region of a spinocerebellar ataxia type 7 gene wherein individuals at risk for developing spinocerebellar ataxia type 7 typically have at least about 30, more typically at least about 37 and even more typically at least about 38 CAG repeats. A person not at risk typically has less than about 19, more typically less than about 15, and even more typically less than about 5 CAG repeats. The methods can include the steps of performing a polymerase chain reaction with oligonucleotide primers capable of amplifying the CAG repeat region located within the spinocerebellar ataxia type 7 gene, and detecting amplified DNA fragments containing the CAG repeat region. The oligonucleotide primers can be selected from the nucleotide region of SEQ ID NO: 9 and from the region of SEQ ID NO: 10. Preferred oligonucleotide primers are SEQ ID NO: 5 and SEQ ID NO: 6.
The methods for identifying individuals at risk for developing spinocerebellar ataxia type 7 can also include detecting the presence of a DNA molecule containing a CAG repeat region of the SCA7 gene by probing genomic DNA digested with a restriction endonuclease and probing the DNA fragments under hybridizing conditions with a detectably labeled gene probe so as to detect a nucleic acid molecule containing a CAG repeat region of an isolated SCA7 gene.
The present invention provides isolated nucleic acids encoding the human SCA7 protein and portions thereof, and isolated proteins and portions thereof encoded from the nucleic acid. The invention also relates to isolated DNA fragments, vectors and isolated recombinant vectors containing the nucleic acids of this invention, oligonucleotide probes and primers that hybridize the SCA7 nucleic acid, host cells transformed or transfected with SCA7 or fragments thereof, compositions containing antibodies that specifically bind to polypeptides encoded by all or part of the SCA7 nucleic acid, a method for detecting the SCA7 disorder including using a biological sample to form an antibody-antigen complex, and to model systems that express the SCA7 protein.
The present invention provides for a kit for detecting whether or not an individual is at risk for developing spinocerebellar ataxia type 7. One preferred kit includes oligonucleotides selected from the nucleotide region of SEQ ID NO: 9 and from the region of SEQ ID NO: 10.
In another aspect of this invention, the invention relates to a procedure for rapidly identifying and isolating expanded repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA using a process of Repeat Analysis, Pooled Isolation, and Detection of individual clones containing expanded repeats (RAPID cloning). The method includes the steps of fractionating a population of DNA fragments and detecting the fraction that contains an expanded repeat, cloning the DNA fragments contained in the fraction of DNA that contains an expanded repeat, and identifying the clones that contain the expanded repeat. The fractionation step can include digesting genomic DNA with a restriction enzyme to obtain DNA fragments, resolving the DNA fragments by gel electrophoresis, dividing into fractions on the basis of size, and detecting the presence of an expanded repeat in each size fraction. The nucleotide sequence flanking the expanded repeat can then be determined and used to design a PCR assay to determine if a particular repeat cosegregates with a given disease.
The invention also relates to an improvement of the repeat expansion detection assay where the rate of temperature change from the denaturation temperature is decreased and wherein the ligation buffer contains formamide. Preferably, the rate of temperature change from the denaturation temperature is decreased to 2 seconds per degree and the ligation buffer contains 4% formamide.
REFERENCES:
patent: 4683194 (1987-07-01), Saiki et al.
patent: 4683195 (1987-07-01), Mullis et al.
patent: 5741645 (1998-04-01), Orr et al.
patent: 5834183 (1998-11-01), Orr et al.
patent: 95/01437 (1995-01-01), None
patent: 97/42314 (1997-11-01), None
Genome Research 6:965-971, Oct. 1996.*
American-Journal of Human Genetics 59 (4 Supple): PA269, Meeting Abstract Oct-Nov. 1996.*
Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1993, pp. 11.2-11.4.*
Benomar et al., “The gene for autosomal dominant cerebellar ataxia with pigmentary macular dystrophy maps to chromosome 3p 12p21.1,”Nature Genetics,10, 84-88 (1995).
David et al., “Th
Benzow Kellie A.
Koob Michael D.
Moseley-Alldredge Melinda L.
Ranum Laura P. W.
Mueting Raasch & Gebhardt, P.A.
Regents of the University of Minnesota
Schwartzman Robert A.
Wang Andrew
LandOfFree
SCA7 gene and method of use does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with SCA7 gene and method of use, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and SCA7 gene and method of use will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2463948