Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-18
2001-06-26
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024330, C536S024300
Reexamination Certificate
active
06251589
ABSTRACT:
This application claims the benefit under 35 U.S.C. §371 of prior PCT International Application No. PCT/JP96/01999 which has an International filing date of Jul. 18, 1996 which designated the United States of America, the entire contents of which are hereby incorporated by reference.
TECHNICAL FIELD
The present invention relates to a method for diagnosing spinocerebellar ataxia type 2 (hereinafter also referred to as “SCA2”) and primers therefor.
BACKGROUND ART
SCA2 is an autosomal dominant, neurodegenerative disorder that affects the cerebellum and other areas of the central nervous system.
It has recently been discovered that the causative genes of five neurodegenerative diseases including dentatorubral-pallidoluysian atrophy (DRPLA) have more CAG repeats than the normal genes. That is, the numbers of CAG repeats in the causative genes of the neurodegenerative diseases are 37 to 100, while those in the normal genes are less than 35.
It has been suggested that the causative gene of SCA2 has an increased number of CAG repeats (Trottier, Y. et al.
Nature,
378, 403-406 (1995)). However, since the causative gene of SCA2 has not been identified, and since its nucleotide sequence has not been determined, SCA2 cannot be diagnosed by a genetic assay.
DISCLOSURE OF THE INVENTION
Accordingly, an object of the present invention is to provide a method for diagnosing SCA2 by genetic assay and to provide primers therefor.
The present invention intensively studied to discover after carrying out Southern blot analysis using a
32
P-labeled single-stranded DNA probe containing 55 CAG repeats (this probe is hereinafter also referred to as “(CAG)
55
probe” (SEQ ID NO:17) on the genomic DNA fragments obtained by digesting the genomic DNAs of SCA2 patients and normal individuals, that a TspEI fragment with a size of 2.5 kb is detected only in SCA2 patients. The (CAG)
55
probe selectively partially hybridizes with a DNA region having not less than about 35 CAG repeats by appropriately selecting the hybridization conditions. The 2.5 kb TspEI fragment was cloned and sequenced. On the other hand, by carrying out Southern blot analysis on the genomic DNAs of normal individuals using the cloned DNA fragment as a probe, normal genes were obtained and sequenced. Comparison between the genes from the SCA2 patients and the genes from the normal individuals revealed that the numbers of the CAG repeats were different and the other portions were substantially the same. Primers were selected such that the CAG repeats are interposed therebetween and PCR was carried out. By measuring the size of the PCR product, the number of CAG repeats in the PCR product was determined. The number of the CAG repeats was measured for 34 chromosomes from SCA2 patients and 286 chromosomes from normal individuals. As a result, in all of the SCA2 genes, the numbers of the CAG repeats were not less than 35, while in normal genes, there were 15 to 24. Therefore, by measuring the number of CAG repeats, the genetic diagnosis of SCA2 can be attained.
That is, the present invention provides a method for diagnosing spinocerebellar ataxia type 2, comprising carrying out PCR using a first primer which hybridizes with a part of the nucleotide sequence shown in SEQ ID NO:1, a second primer which hybridizes with a part of the nucleotide sequence shown in SEQ ID NO:3, and a test DNA as a template, and measuring the number of CAG repeats in the amplified PCR product. The present invention also provides the above-mentioned first and second primers which are used for the above-described method.
By the present invention, genetic diagnosis of SCA2 was first accomplished. Therefore, it is expected that the present invention will greatly contribute to the diagnosis of SCA2.
REFERENCES:
patent: 5853995 (1998-12-01), Lee
Pulst, “Anticipation in spinocerebellar ataxia type 2”, Sep. 1993, Nature Genetics, vol. 5, pp. 8-10.*
Filla et al., “Has spinocerebellar ataxia type 2 a distinct phenotype?”, Apr. 1995, Neurology, 45, pp. 793-796.*
Gispert et al., “Chromosomal assignment of the second locus for autosomal dominant cerebellar ataixa (SCA2) to chromosome 12q23-24.1”, Jul. 1993, Nature Genetics, vol. 4, pp. 295-299.*
Yamagata et al., Jpn. J. Electroph., vol. 38(6), abstract, 1994.*
B. T. Teh et al.,Am. J. Hum. Genet., vol. 56, pp. 1443-1449 (1995).
Y. Trottier et al.,Nature, vol. 378, pp. 403-406 (1995).
Koide et al.,Nature Genetics, vol. 6, pp. 9-13, (Jan. 1994).
Sanpei et al.,Biochemical and Biophysical Research Communications, vol. 212, No. 2, pp. 341-346, (Jul. 1995).
Sanpei Kazuhiro
Tsuji Shoji
Arthur Lisa B.
Birch & Stewart Kolasch & Birch, LLP
Goldberg Jeanine
SRL, Inc.
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