CA2232311A1 - Method for diagnosis of spinocerebellar ataxia type 2 and primers therefor - Google Patents
Method for diagnosis of spinocerebellar ataxia type 2 and primers therefor Download PDFInfo
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Abstract
A method for specifically diagnosing spinocerebellar ataxia type 2 (SCA2). The method comprises effecting PCR by employing the DNA to be tested as a template and using a first primer comprising a nucleic acid which is hybridizable with a part of the base sequence represented by SEQ ID NO.1 in the Sequence Listing and a second primer comprising a nucleic acid which is hybridizable with a part of the base sequence represented by SEQ ID NO.2 in the Sequence Listing and determining the number of CAG repeat units in the PCR product thus amplified. In this method, the gene of a patient with SCA2 has the number of CAG repeat units of 35 or above while the gene of a normal subject has the number of from 15 to 24, which enables the diagnosis of SCA2.
Description
98- 3 - 1 6 i 1 9: 33 ;TA~ I GAWA PATENTcA 022323ll 1998-03-l7 ; 0332389 1 83 # 2/ 2 6 SPECIF:[CATION
Method for Diagnosing Spinocerebellar Ataxia Type 2 and Primers Therefor TECHNICA~ 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 ce.rebellum 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 (~R~LA) 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 t:hat 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 ldentified, and since its nucleotide sequence has not been determined, SCA2 cannot be diagnosed by a genetic assay.
DISCLOSURE OF THE INV~NTION
Accordingly, an ob~ect of the present invention is to provide a method for diagnoslng SCA2 by genetic assay 9~- 3-1 6; 1 9: 33 ;TAN I GA\~A PATENTcA 02232311 1998-03-17 ; 0332389 1 83 # ~/ '7~3 and to provide primers therefor.
The present inventors intensively studied to discover after carrying out Southern blot analysis using a 32P-labeled single-strandecl ~NA probe containing 55 CAG
repeats (this probe is hereinafter also referred to as "(CAG)5s probe") on the genomic DNA fragments obtained by digesting the genomic DNAs of SCA2 patients and normal individuals, that a Tsp~I fragment with a size of 2.5 kb is de~ected only in SCA2 patients. The (CAG) 55 probe selectively partially hybridizes with a DNA region having not less than about 35 CA~ 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.
Co~parison between the geneC from the SCA2 patients and the genes from the normal individuals revealed that the numbers of the CA~ 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 num~er 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 o~ the 98-- 3--1 6; 1 9: 33 : TAN I GAWA PATENT S~a r t 3 ; 0332389 1 83 # 4/ 2 6 CAG repeats were not less than 35, while in normal genes, they 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 a.taxia type 2, comprising carrying out PCR using a first primer which hybridizes with a part of the nucleotice sequence shown in SEQ ID
NO:1, a second primer which hybrid]zes with a part of the nucleotide sequence shown ln SEQ ID NO:2, and a test DNA
as a template, and measuring the number of CAG repeats in the amplified PCR product. rhe present invention also provides the abo~e-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 lnvention will greatly contribute to the diagnosis of SCA2.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the nucleotide sequence of the causative gene of SCA2;
Fig. 2 is a pedigree chart of the SCA2 patients who donated the genomic DNAs used in the Examples;
Fig. 3 is a restriction map of Tsp-2 which is a genomlc DNA fragment origina.ted from a normal allele;
Fig. 4 shows the nucleotide sequence of Tsp-2 together with the regions with which each primer 98-- 3--1 6; ~ 9: 33 ;TAI'I I GAWA PATENTcA 02232311 1998-03- 17 ; 0332389 1 8. # 5 / 2 6 hybridizes;
Fig. 5 shows nucleotide sequences of the region of the CAG repeats and of the region immediately thereafter;
Fig. 6 shows the distribution of the numbers of the CAG repeat units in the normal and SCA2 genes, which were measured by the method according to the present invention; and Fig. 7 shows the relationship between the number of CAG repeat units and the age of onset of SCA2.
BEST MODE FOR CARRYI:NG OUT THE INVENTION
As will ~e described in detail in the Examples below, the partial nucleotide sequences of the causative genes of SCA2 were determined by the present invention, and the partial nucleotide sequences of the corresponding genes of normal individuals were also determined. Comparison between the SCA2 causative genes and the normal genes revealed that the numbers of CAG triplet repeats in the genes are different. More p.~rticularly, as will be described in detail in the Examples below, the SCA2 genes have 35 to 55 CAG repeat units while in normal lndividuals, ~he numbers of the CAG repeats were 15 to 24, and 94.4% of the normal individuals had 22 CAG repeats.
Thus, it was proved that SCA2 can be diagnosed by measuring the number of the CAG repeat units.
The nucleotide sequence of the gene participating in the onset of SCA2, which was determined by the present invention, is shown in Fig. 1. In Fig. 1, the region 98-- 3-1 6; 1 ~: 33 i TAN I GAWA PATENT Sma r t 3 ; 0332389 1 83 # 6/ 2 6 ~j indicated by ~CAG) n is the region of the CAG triplet repeats. The region of (CCG CCG CAG) may or may not be contained in the SCA2 gene, and is not contained in the normal gene. The region shown in uppercase letters is the region of which sequence was determined from both genomic DNA and cDNA, and the regions shown in lowercase letters are the regions of which sequences were determined from cDNA. The sites at which the base is different in the genomic DNA and the cDNA are shown in two layers and the upper character indicate the base determined from the cDNA.
As will be described in detail in the Examples below, although there are some other minor di~ferences, the main difference between the SCA2 gene and the normal gene resides in the number of CAG repeat units shown as ~CAG) n in Fig. 1. Thus, by measuring the number of the CAG
repeat units, SCA2 can be diagnosed.
In the method of the present invention, to measure the number of the CAG repeat unitsl the region including the CAG repeat region is amplified by PCR. This can be accomplished by carrying out the PCR using primers hybridizing with the regions upstream and downstream of the CAG repeat region, respectively.
The nucleotide sequence of the region upstream of the CAG repeat region is shown in SEQ ID NO:1 in the SEQUENCE LISTING, and that of the region downstream of the CAG repeat region is shown in SEQ ID NO:2. PCR is 98-- 3--1 6: 1 9: 33 ; TAN GAWA PATI~NT Sma r t 3 : ~332389 1 83 # 7/ " ~
performed using oligonucleotide primers which hybridize with the sense chain and the antisense chain of the regions in the upstream and downstream regions of the CAG
repeat region, respecti~ely. Any primers which hybridize with any part of the above-mentioned regions may be employed. The size of the primer is not restricted and usually 15 to 50 nucleotides, preferably 1~ to 25 nucleotides.
Using these primers, and using a test DNA as the template, PCR ls performed. The test DNA is the DNA
sampled from a person to be diagnosed for SCA~, and may be prepared from peripheral bIood or the like by a conventional method. In cases where the primers which hybridize with the regions upstream and downstream of the CAG repeat region, respecti~ely, which regions are shown in uppercase letters in Fig 1, are employed, not only genomic DNA, but also cDNA may be used as the template.
Since the PCR method per se is well-known in the art and a kit therefor is commercia~Lly a~ailable, PCR can be easily carried out. The details of the PCR are also described in the Examples below.
Then the number of the CAG repeat units in the amplified PCR product is measured. This may be carried out by, ~or example, sequencing the PCR product.
Alternatively, this may be ,-arried out by performing gel electrophoresis toge~her with appropriate markers and determining the size of the PCR product. In the Examples 98-- 3-1 6; 1 9: 33 ; TAN I GAWA PATENT Sma r t 3 ; 03323891 83 # 8/ ~ 6 described below, gel electrophoresis using SCA2 genes having slightly varying numbers of CAG repeat units as size markers was carried out so as to determine the size of the PCR product and, in t~]rn, the number of the CAG
repeat units contained therein. It should be noted that the methods for measuring the number of the CA~ repeat units in the PCR product are not restrlcted to the above-mentioned methods, and any method which can determine the number of the CAG repeat units may be employed. Further, a method which can only distinguish whether the number of CAG repeat units is about not less than 35 or not may also be employed. Such a melhod is also included in the method for "measuring the number of the CAG repeat units"
in the context of the present invention. For example, a probe such as a ~CAG~ss probe mentioned above may be subjected to hybridization with the ~CR product in a condition under which it hybridizes only with the DNAs h~ving not less than about 35 CAG repeat units.
Examples The present invention will now be described in more concretely by way of examples. It should be understood, however, the examples are presented for the illustration purpose only and should not be interpreted in any restrictive way.
Reference Example 1 Preparation of (CAG) 55 Probe A genomic DNA segment of 3RPLA gene containing a CAG
repeat with 55 repeat units was amplified from the 98-- 3--1 ~; 1 9: 33 : TAN I GAWA PATEI'IT Sma r t 3 ; 0332389 1 83 # 9,' 2 6 genomic ~NA of a patient with ~RPLA tKoide, R. et al., Nature Genet., 6, g-13 ~1994)) and was subcloned into a plasmid vector, pT7~1ue T (p-2~93) . The p-2093 plasmid contains the (CAG)55 and the flanking sequences. That is, the plasmid contains the sequence of 5'-CAC CAC CAG CAA
CAG CAA (CAG)55 CAT CAC GGA ~C TCT GGG CC-3'. Using a pair of oligonucleotides 5'-CAC CAC CAG CAA CAG CAA CA-3'and 5'-biotin-GGC CCA G~G 'rTT ~CG TGA TG-3', PCR was performed in a total volume of 16 ul containing 10 mM
Tris~HCl, pH8.3, 50 mM KCl, 1.5 mM MgCl2, 2M N,N,N-trimethylglycine, 0.1 mM TTP, 0.1 Mm dCTP, 0.1 mM dGTP, 9 25 MBq of l~-32P~dATP (222 TBq/mmol), 0.5 mM each of the two primers, 0.3 ng of plasmid DNA (p-2093) and 2.0 U
of Taq DNA polymerase (Takara Shuzo, Kyoto, Japan).
After an initial 2-min. denaturation at 94~C, ~CR was performed for 30 cycles consisting of 1-min. denaturation at 94~C, 1-min. annealing at 54~C and 3-min. extension at 72~C, followed by a final extension at 72~C for 10 min.
A single-stranded (CAG)~5 prcbe was isolated using streptavidln-coated magnetic beads ~Dynabeads M-280, Streptavidin;Dynal AS, Oslo, Norway). That is, after washing of the PCR products immobilized on the magnetic beads with 90 ~l of a solution containing 5 ~M Tris-HCl (pH 7.5), 0.5 mM EDTA and 1 M NaC1, the non-biotinylated strand contalnlng the radio-label was separated from the biotinylated strand by incubation in 5C ul of 0.1 M NaOff for 10 min. The resultant supernatant was directly added 3-1 6; 1 9: 33 ;TAN GAWA PATENTcA 02232311 1998-03-17 ; ()3323891 83 # 1 0/ 26 to the hybridization solution described below.
Incidentally, using the single-stranded ~CAG) 55 probe prepared as described above, Southern blot analysis was carried out on the androgen receptor genes containing 9, 22, 43 and 51 CAG repeat units, respectively. As a result, the (CAG) 55 probe strongly hybridized with the genes ha~ing 43 and 51 CAG repeats units, respectively, but scarcely hybridized with the gene having 22 CAG
repeat units, and did not hybridize at all with the gene having 9 CAG repeat units (K. Sanpei et al., Biochemical and Biophysical Research Communications, Vol.212, No.2, 1995, pp.341-346~. Thus, by using this probe, hybridization may be selectively attained only with DNAs containing a number of (e.g., 35 or more) CAG repe2t units if the hybridization conditions are appropriately selected.
~xample 1 Determination of Nucleo~ide Sequence of SCA2 Gene Fig. 2 shows a pedigree chart of SCA2 patients. In this pedigree chart, males are represented by squares and females are represented by circles. SCA2 patients are represented by black squares or circles, and unaffected persons are represented by white squares or circles.
High-molecular-weight g-enomic DNA (15 ~g~ was digested with 100 U of TspEI: (Toyobo, Osaka, Japan), electrophoresed through l.C~i agarose gels and transferred to nitrocellulose membranes. The membranes were 98- 3-16i19:33 ;TANlGAwA PATENTCA 022323l1 1998-03-l7 ; 0332389183 # 11/ 26 hybridized with the (CAG) 55 probe described above.
Hybridization was performed in a solution containing 2.75 x SS~E (1 x SSPE=150 mM NaCl, 10 mM NaHzPO4, 1 mM EDTA), 50% formamide, 5 x Denhardt"~ solution, 100 ng/ml of sheared salmon sperm DNA and the (CAG) 55 probe (6 x 106 cpm/ml) at 62~C for 1~ hours. After the hybridization, the membranes were washed with 1 x SSC (150 mM NaCl, 15 ~M sodium citrate) containing 0.5~ SDS at 65~C for ~.5 hours. The membranes were autoradiographed for 16 hours to Kodak Bio Max MS film at -70~C using an MS
intensifying screen.
As a result, 2.5 kbp TspEI fragment hybridized with the probe was detected only in all of the SCA2 patients.
&enomic DNA (270 ~g) from an SCA2 patient (indi~idual 7 in Fig. 2) was digested by TspEI and subjected to agarose gel electrophoresis. Genomic ~NA
fragments including the 2.5 kb TspEI fragment were cloned into an EcoRI-cleaved ~ZAPII vector. The genomic library was screened using the (CA~) 55 probe under the hybridization condition described above. A genomic clone, Tsp-l, containing an expanded CAG repeat was isolated.
After remo~al of the probe, the above-described genomic library was screenecl again using the Tsp-l as a probe, which was labeled by the random priming.
Hybridization was carried out in a solution containing 5 x SSC, 1 x Denhardt's solution, 10~ dextran sulfate, 20 mM sodium phosphate, 400 ~ug,/ml human placental DNA and 98-- 3-1 6: 1 9: 33 i TAN I GAWA PATENT Sma r t 3 : ~332389 1 83 # 1 2/ ~ ~
the Tsp-l probe at 42~C for 18 hours. After the hybridization, the membranes were washed finally in O.l x SSC - 0.1% SDS at 52~C for 0.5 hours. The membranes were autoradiographed for 24 hours to Kodak Bio Max MS films at -70~C using an MS intensif-ying screen. As a result, a genomic clone, ~sp-2, originated from a normal allele was isolated.
A restriction map of Tsp-2 is shown in Fig. 3. The CAG repeat (open box) is located in the putative first exon (solid box).
Nucleotide sequence of the SmaI-ApaI fragment (630 bp) of Tsp-2 was determined. The sequence is shown in Fig. 4 and in SEQ ID NO:3 in the SEQUENCE LISTING. In Fig. 4, the region of which sequence was determined from both the genomic DNA and cDNA (described below) is shown in uppercase letters and the regions of which sequences were determined from the cDNA is shown in lowercase letters. The sites at which the base is different in the genomic D~A and the cDNA are shown in two ~ayers and the upper character indicate the base determined from the cDNA. The deduced amino acid sequence is shown below the nucleotide sequence. Translation initiated from the ATG
codon in the longest open reading frame in the putative first exon.
A human cerebral cortex cDNA library (Clonetech, Palo Alto, CA, USA~ was screened using oligonucleotides, F-l a~d R-l, designed to flank the CAG repeat as shown in 98- 3-16:19:33 ;TANIGAWA PATENT Sma~t3 ;0332389183 # 13,' 26 ~ig. 4, as the probes. Hybridization was performed for 18 hours at 55~C in a solution containing 6 x SSC, 10 x Denhardt's solution, 0.5% SDS, 0.05% sodium pyrophosphate, lOQ ng/ml sheared salmon sperm DNA and the end-labeled oligonucleotide probe. After the hybridization, the membranes were washed finally in 6 x SSC containing 0.5%
SDS and 0.05~ sodium pyrophosphate for 0.5 hours at 55~C.
A clone, FC-1, which gave a hybridization signal with both the oligonucleotide probes, was obtained. To identify the 5' end of the c~)NA, 5'-RACE was performed using 5'-RACE-Ready cDNA ~Clonetech, Palo Alto, CA, USA).
The primer R-1 was used in the primary PCR and the primer R-2 ~see Fig. 4) was used in the secondary ~CR. As the forward primer, F-1 (see Fig. 4) was used in both the primary and secondary PCR. The 5'-RACE product, 350 bp in size, was subcloned into a pT7Blue T vector (5 '~RACE-1). The identity of 5'-RACE--l was confirmed by the overlap of the nucleotide sequence with those of Fc-1, Tsp-l and Tsp-2. Nucleotide sequences were determined by the dideoxynucleotide chain termination method using double-stranded plasmid ~NAs as the templates.
PCR products obtained from genomic DNA of normal and SCA2-affected individuals using the primer pair of F-1 (forward side) and R-1 (reverse side) were cloned i~to a plasmid vector, pT7Blue, followed by determination of the nucleotide sequence. The results are shown in Fig. 5.
As shown in Fig. 5, the norma~ genes have one or two 9 8-- 3--1 6: 1 9: 3 3 ; TAN I GAWA P AT E N T CA 022323 1 1 1998 - 03 - 17 ; o 3 3 2 3 8 ~3 1 8 .: # 1 4 / 2 6 CAA, while SCA2 genes do not. Further/ in SCA2 genes, CCG CCG CAG may be inserted into the site immediately after the CAG repeat region.
Example 2 Measurement of CAG Repeat ~nits in Sample Numbers of CAG repeat units were determined by polyacrylamide gel electrophoresis analysis of PCR
products obtained using the primer pair cf F-1 and R-1.
PCR was performed in a total volume of 10 ~l containing 10 mM Tris-HCl, pH 8.3, 50 ~ KCl, 2.0 mM MgCl2, 1.7 M
N,N,N-trimethylgly~ine, lllK3q of [Q-32P]dCTP (111 Tbq/mmol), 30 ~M dCTP, and 200 ,uM each of dATP, dGTP and TTP, 0.25 ~M each of the two primers, 200 ng of genomic DNA and 1.25 U of Taq DNA polymerase. After an initial 2-min denaturation at 95~C, T'CR was performed for 32 cycles of 1-min denaturation at 95~C, 1-min annealing at 60~C and 1-min extension a~ 72~C, followed by a final extension at 72~C for 5 min. Sequence ladders obtained using the cloned genomic segments of the SCA2 gene, which contain various sizes of CAG repeats, were used as size markers. For normal alleles containing one or two CAA
interruptions, the numbers of the CAA units were included in the CAG repeat size. For SCA2 alleles having expanded CAG region, the above-menticned insert sequence immediately after the CAG region was not included in the size of the CAG region.
By the above-described method, the numbers of the CAG repeat units of normal individuals (286 chromosomes) 98- 3-16;19:33 ;TANIGAWA PATENTcA 02232311 1998-03-17 : n3323~918.~ # 15/ 2 and 10 pedigrees of SCA2 patients (34 SCA2 chromosomes) were determined. The results are shown in Fig. ~. In Fig. 6, open bars indicate the results of the normal genes and solid bars indicate the results of the SCA2 genes.
As is apparent from Fig.. 6, in all of the normal genes, the numbers of the CAG repeat units were not more than 24, while in all of the SCA2 genes, they were not less than 35. Thus, it was proved that genetic diagnosis of SCA2 can be attained by the method of the present invention.
The relationship betwee:n the number of the CAG
repeat units determined by the method described above and the age at onset of symptoms of SCA2 is shown in Fig. 7.
An inverse correlation was observed statistically. This suggests that expansion of the number of the CAG repeat units relates to the onset cf symptoms of SCA2.
INDUSTRIAL AVAILABILITY
By the present inventicn, genetic assay of SCA2 was first attained. Thus, it is expected that the present invention will greatly contribute ~o the diagnosis of SCA2.
98- 3--1 6; 1 9: 33 ; TAN I GAWA PATENT Sma r t 3 : 0332389 1 8~ # ' 6,' 2 5 SEQUENC~ LISTING
SEQ ID NO:1 SEQUENCE LENGTH: 355 SEQUENCE TYPE: nucleic acid STRNDEDNESS: double S~QUENCE DESCRIPTION
GGGACCGTAT CCCTCCGCCG CCCCTCCCCC GCCCGGCCCC GGCCCCCCTC CCTCCC~GCA 60 GAGCTCGGCT CCCTCCGCCT CAGACTGTTT TGGTA&CAAC GGCAACGGCG GCGGCGCGTT 120 Met Ser Leu Lys Pro SEQ ID NO:2 S~QUENCE LENGTH: 205 SEQUENCE TYPE: nucleic acid STRNDEDNESS: double SEQUENCE DESCRIPTION
CCG CC& CCC GCG GCT GCC AAT GTC CGC AAG CCC GGC GGC AGC GGC CTT 48 Pro Pro Pro Ala Ala Ala Asn Val Arg Lys Pro Gly Gly Ser Gly Leu Leu Ala Ser Pro Ala Ala Ala Pro Ser Pro Ser Ser Ser Ser Val Ser 98-- 3--1 6; 1 ~: 33 ;TAN I GAWA PATENT Sma r t3 : 03323891 8. # 1 7 ~ 2 5 CA 022323ll lsg8-03-l7 Ser Ser Ser Ala Thr Ala Pro Ser Ser Val Val Ala Ala Thr Ser Gly GGC GGG AGG CCC GGC CTG GGC AGGTGGGT~T CGGCACCCCA GCCCCCCTCC 19 Gly Gly Arg Pro Gly Leu Gly SEQ ID NO:3 SEQUENCE LENGTH: 623 SEQUENC~ TYPE: nucleic acid STRNDEDNESS: double SEQUENCE D~SCRI~TION
Met Ser Leu Lys Pro Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln lO 15 20 CA& CAG CAG CAG CAG CCG CCG CCC GCG GCT GCC M T GTC CGC AAG CCC 451 Gln Gln Gln Gln Gln Pro Pro Pro Ala Ala Ala Asn Val Arg Lys Pro 98- 3-1 6; 1 9: 33 ; TAN I GAWA PATENT Sma rt 3 ~ 03323891 8'-: # 1 8,/ 2~
Gly Gly Ser Gly Le~ Leu Ala Ser Pro Ala Ala Ala Pro Ser Pro Ser Ser S~r Ser Val Ser Ser Ser Ser Ala Thr Ala Pro Ser Ser Val Val ~5 60 65 GCG GCG ACC TCC GGC GGC GGG AGG CCC GGC CTG BGC AGGTG>GT ~93 Ala Ala Thr Ser Gly Gly Gly Arg Pro Gly Leu Gly SEQ ID N0:4 SEQUENCE LENGTH: 213 SEQUENCE TYPE: nucleic acid STRNDEDNESS: single SEQUENCE DESCRIPTION
CAGCAGCAGC A&CAGCA&CA GCAGCAGCAG CAGCAGCAGC AGCAGCAGCA GCAGCAGCAG 120 SEQ ID NO:5 SEQUENCE L~NGTH: 20 SEQUENCE TYPE: nucleic acid STRNDEDNESS: single SEQUENCE DESCRIPTION
98- 3-1 6: 1 9: 33 ;TAN I ~AWA PATENT Srra -t3 ; 03323891 83 # 1 9.' 26 SEQ ID NO:6 SEQUENCE LENGTH: 20 SEQ~ENCE TYPE: nucleic acid SEQUENC~ DESCRIPTION
SEQ ID NO:7 SEQUENCE LENGTH: 75 SEQUENCE TYPE: nuclelc acid SE~JENCE DESCRIPTION
SEQ ID NO:8 SEQUENCE LENGTH: 78 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION
CAGCAGCAGC AGCA&CAGCA GCAGCAACAG CAGC M CAGC AACAGCAGCA GCAGCAGCAG 60 SEQ ID NO:9 SEQUEN~E LENGTH: 78 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION
CAGCAGCAGC AGCAGCAGCA GCAGCAGCAG C M CAGCA&C M CAGCAGCA GCAGCAGCAG 60 98- 3-1 6; 1 9: 33 ; TAN I GAWA PATENT Sma r t 3 ; 03323891 83 # 2 0,' 2 1~
SEQ ID NO:10 SEQUENCE LENGTH: 78 SEQUENCE TYP~: nucleic acid SEQUENCE DESCRIPTION
SEQ ID NO:11 SEQUENCE LE~GTH: 78 SEQUENCE TY~E: nucleic acid SEQUENCE DESCRIPTION
CAGCAGCAGC AGCAGCAGCA GC M CAGCAG CAGCAGCAGC AACAGCAGCA &CAGCAGCCG 60 SEQ ID NO:12 SEQUENCE LENGTH: 78 SEQUENCE TYPE: nucLeic acid SEQUENCE DESCRIPTION
Method for Diagnosing Spinocerebellar Ataxia Type 2 and Primers Therefor TECHNICA~ 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 ce.rebellum 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 (~R~LA) 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 t:hat 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 ldentified, and since its nucleotide sequence has not been determined, SCA2 cannot be diagnosed by a genetic assay.
DISCLOSURE OF THE INV~NTION
Accordingly, an ob~ect of the present invention is to provide a method for diagnoslng SCA2 by genetic assay 9~- 3-1 6; 1 9: 33 ;TAN I GA\~A PATENTcA 02232311 1998-03-17 ; 0332389 1 83 # ~/ '7~3 and to provide primers therefor.
The present inventors intensively studied to discover after carrying out Southern blot analysis using a 32P-labeled single-strandecl ~NA probe containing 55 CAG
repeats (this probe is hereinafter also referred to as "(CAG)5s probe") on the genomic DNA fragments obtained by digesting the genomic DNAs of SCA2 patients and normal individuals, that a Tsp~I fragment with a size of 2.5 kb is de~ected only in SCA2 patients. The (CAG) 55 probe selectively partially hybridizes with a DNA region having not less than about 35 CA~ 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.
Co~parison between the geneC from the SCA2 patients and the genes from the normal individuals revealed that the numbers of the CA~ 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 num~er 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 o~ the 98-- 3--1 6; 1 9: 33 : TAN I GAWA PATENT S~a r t 3 ; 0332389 1 83 # 4/ 2 6 CAG repeats were not less than 35, while in normal genes, they 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 a.taxia type 2, comprising carrying out PCR using a first primer which hybridizes with a part of the nucleotice sequence shown in SEQ ID
NO:1, a second primer which hybrid]zes with a part of the nucleotide sequence shown ln SEQ ID NO:2, and a test DNA
as a template, and measuring the number of CAG repeats in the amplified PCR product. rhe present invention also provides the abo~e-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 lnvention will greatly contribute to the diagnosis of SCA2.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the nucleotide sequence of the causative gene of SCA2;
Fig. 2 is a pedigree chart of the SCA2 patients who donated the genomic DNAs used in the Examples;
Fig. 3 is a restriction map of Tsp-2 which is a genomlc DNA fragment origina.ted from a normal allele;
Fig. 4 shows the nucleotide sequence of Tsp-2 together with the regions with which each primer 98-- 3--1 6; ~ 9: 33 ;TAI'I I GAWA PATENTcA 02232311 1998-03- 17 ; 0332389 1 8. # 5 / 2 6 hybridizes;
Fig. 5 shows nucleotide sequences of the region of the CAG repeats and of the region immediately thereafter;
Fig. 6 shows the distribution of the numbers of the CAG repeat units in the normal and SCA2 genes, which were measured by the method according to the present invention; and Fig. 7 shows the relationship between the number of CAG repeat units and the age of onset of SCA2.
BEST MODE FOR CARRYI:NG OUT THE INVENTION
As will ~e described in detail in the Examples below, the partial nucleotide sequences of the causative genes of SCA2 were determined by the present invention, and the partial nucleotide sequences of the corresponding genes of normal individuals were also determined. Comparison between the SCA2 causative genes and the normal genes revealed that the numbers of CAG triplet repeats in the genes are different. More p.~rticularly, as will be described in detail in the Examples below, the SCA2 genes have 35 to 55 CAG repeat units while in normal lndividuals, ~he numbers of the CAG repeats were 15 to 24, and 94.4% of the normal individuals had 22 CAG repeats.
Thus, it was proved that SCA2 can be diagnosed by measuring the number of the CAG repeat units.
The nucleotide sequence of the gene participating in the onset of SCA2, which was determined by the present invention, is shown in Fig. 1. In Fig. 1, the region 98-- 3-1 6; 1 ~: 33 i TAN I GAWA PATENT Sma r t 3 ; 0332389 1 83 # 6/ 2 6 ~j indicated by ~CAG) n is the region of the CAG triplet repeats. The region of (CCG CCG CAG) may or may not be contained in the SCA2 gene, and is not contained in the normal gene. The region shown in uppercase letters is the region of which sequence was determined from both genomic DNA and cDNA, and the regions shown in lowercase letters are the regions of which sequences were determined from cDNA. The sites at which the base is different in the genomic DNA and the cDNA are shown in two layers and the upper character indicate the base determined from the cDNA.
As will be described in detail in the Examples below, although there are some other minor di~ferences, the main difference between the SCA2 gene and the normal gene resides in the number of CAG repeat units shown as ~CAG) n in Fig. 1. Thus, by measuring the number of the CAG
repeat units, SCA2 can be diagnosed.
In the method of the present invention, to measure the number of the CAG repeat unitsl the region including the CAG repeat region is amplified by PCR. This can be accomplished by carrying out the PCR using primers hybridizing with the regions upstream and downstream of the CAG repeat region, respectively.
The nucleotide sequence of the region upstream of the CAG repeat region is shown in SEQ ID NO:1 in the SEQUENCE LISTING, and that of the region downstream of the CAG repeat region is shown in SEQ ID NO:2. PCR is 98-- 3--1 6: 1 9: 33 ; TAN GAWA PATI~NT Sma r t 3 : ~332389 1 83 # 7/ " ~
performed using oligonucleotide primers which hybridize with the sense chain and the antisense chain of the regions in the upstream and downstream regions of the CAG
repeat region, respecti~ely. Any primers which hybridize with any part of the above-mentioned regions may be employed. The size of the primer is not restricted and usually 15 to 50 nucleotides, preferably 1~ to 25 nucleotides.
Using these primers, and using a test DNA as the template, PCR ls performed. The test DNA is the DNA
sampled from a person to be diagnosed for SCA~, and may be prepared from peripheral bIood or the like by a conventional method. In cases where the primers which hybridize with the regions upstream and downstream of the CAG repeat region, respecti~ely, which regions are shown in uppercase letters in Fig 1, are employed, not only genomic DNA, but also cDNA may be used as the template.
Since the PCR method per se is well-known in the art and a kit therefor is commercia~Lly a~ailable, PCR can be easily carried out. The details of the PCR are also described in the Examples below.
Then the number of the CAG repeat units in the amplified PCR product is measured. This may be carried out by, ~or example, sequencing the PCR product.
Alternatively, this may be ,-arried out by performing gel electrophoresis toge~her with appropriate markers and determining the size of the PCR product. In the Examples 98-- 3-1 6; 1 9: 33 ; TAN I GAWA PATENT Sma r t 3 ; 03323891 83 # 8/ ~ 6 described below, gel electrophoresis using SCA2 genes having slightly varying numbers of CAG repeat units as size markers was carried out so as to determine the size of the PCR product and, in t~]rn, the number of the CAG
repeat units contained therein. It should be noted that the methods for measuring the number of the CA~ repeat units in the PCR product are not restrlcted to the above-mentioned methods, and any method which can determine the number of the CAG repeat units may be employed. Further, a method which can only distinguish whether the number of CAG repeat units is about not less than 35 or not may also be employed. Such a melhod is also included in the method for "measuring the number of the CAG repeat units"
in the context of the present invention. For example, a probe such as a ~CAG~ss probe mentioned above may be subjected to hybridization with the ~CR product in a condition under which it hybridizes only with the DNAs h~ving not less than about 35 CAG repeat units.
Examples The present invention will now be described in more concretely by way of examples. It should be understood, however, the examples are presented for the illustration purpose only and should not be interpreted in any restrictive way.
Reference Example 1 Preparation of (CAG) 55 Probe A genomic DNA segment of 3RPLA gene containing a CAG
repeat with 55 repeat units was amplified from the 98-- 3--1 ~; 1 9: 33 : TAN I GAWA PATEI'IT Sma r t 3 ; 0332389 1 83 # 9,' 2 6 genomic ~NA of a patient with ~RPLA tKoide, R. et al., Nature Genet., 6, g-13 ~1994)) and was subcloned into a plasmid vector, pT7~1ue T (p-2~93) . The p-2093 plasmid contains the (CAG)55 and the flanking sequences. That is, the plasmid contains the sequence of 5'-CAC CAC CAG CAA
CAG CAA (CAG)55 CAT CAC GGA ~C TCT GGG CC-3'. Using a pair of oligonucleotides 5'-CAC CAC CAG CAA CAG CAA CA-3'and 5'-biotin-GGC CCA G~G 'rTT ~CG TGA TG-3', PCR was performed in a total volume of 16 ul containing 10 mM
Tris~HCl, pH8.3, 50 mM KCl, 1.5 mM MgCl2, 2M N,N,N-trimethylglycine, 0.1 mM TTP, 0.1 Mm dCTP, 0.1 mM dGTP, 9 25 MBq of l~-32P~dATP (222 TBq/mmol), 0.5 mM each of the two primers, 0.3 ng of plasmid DNA (p-2093) and 2.0 U
of Taq DNA polymerase (Takara Shuzo, Kyoto, Japan).
After an initial 2-min. denaturation at 94~C, ~CR was performed for 30 cycles consisting of 1-min. denaturation at 94~C, 1-min. annealing at 54~C and 3-min. extension at 72~C, followed by a final extension at 72~C for 10 min.
A single-stranded (CAG)~5 prcbe was isolated using streptavidln-coated magnetic beads ~Dynabeads M-280, Streptavidin;Dynal AS, Oslo, Norway). That is, after washing of the PCR products immobilized on the magnetic beads with 90 ~l of a solution containing 5 ~M Tris-HCl (pH 7.5), 0.5 mM EDTA and 1 M NaC1, the non-biotinylated strand contalnlng the radio-label was separated from the biotinylated strand by incubation in 5C ul of 0.1 M NaOff for 10 min. The resultant supernatant was directly added 3-1 6; 1 9: 33 ;TAN GAWA PATENTcA 02232311 1998-03-17 ; ()3323891 83 # 1 0/ 26 to the hybridization solution described below.
Incidentally, using the single-stranded ~CAG) 55 probe prepared as described above, Southern blot analysis was carried out on the androgen receptor genes containing 9, 22, 43 and 51 CAG repeat units, respectively. As a result, the (CAG) 55 probe strongly hybridized with the genes ha~ing 43 and 51 CAG repeats units, respectively, but scarcely hybridized with the gene having 22 CAG
repeat units, and did not hybridize at all with the gene having 9 CAG repeat units (K. Sanpei et al., Biochemical and Biophysical Research Communications, Vol.212, No.2, 1995, pp.341-346~. Thus, by using this probe, hybridization may be selectively attained only with DNAs containing a number of (e.g., 35 or more) CAG repe2t units if the hybridization conditions are appropriately selected.
~xample 1 Determination of Nucleo~ide Sequence of SCA2 Gene Fig. 2 shows a pedigree chart of SCA2 patients. In this pedigree chart, males are represented by squares and females are represented by circles. SCA2 patients are represented by black squares or circles, and unaffected persons are represented by white squares or circles.
High-molecular-weight g-enomic DNA (15 ~g~ was digested with 100 U of TspEI: (Toyobo, Osaka, Japan), electrophoresed through l.C~i agarose gels and transferred to nitrocellulose membranes. The membranes were 98- 3-16i19:33 ;TANlGAwA PATENTCA 022323l1 1998-03-l7 ; 0332389183 # 11/ 26 hybridized with the (CAG) 55 probe described above.
Hybridization was performed in a solution containing 2.75 x SS~E (1 x SSPE=150 mM NaCl, 10 mM NaHzPO4, 1 mM EDTA), 50% formamide, 5 x Denhardt"~ solution, 100 ng/ml of sheared salmon sperm DNA and the (CAG) 55 probe (6 x 106 cpm/ml) at 62~C for 1~ hours. After the hybridization, the membranes were washed with 1 x SSC (150 mM NaCl, 15 ~M sodium citrate) containing 0.5~ SDS at 65~C for ~.5 hours. The membranes were autoradiographed for 16 hours to Kodak Bio Max MS film at -70~C using an MS
intensifying screen.
As a result, 2.5 kbp TspEI fragment hybridized with the probe was detected only in all of the SCA2 patients.
&enomic DNA (270 ~g) from an SCA2 patient (indi~idual 7 in Fig. 2) was digested by TspEI and subjected to agarose gel electrophoresis. Genomic ~NA
fragments including the 2.5 kb TspEI fragment were cloned into an EcoRI-cleaved ~ZAPII vector. The genomic library was screened using the (CA~) 55 probe under the hybridization condition described above. A genomic clone, Tsp-l, containing an expanded CAG repeat was isolated.
After remo~al of the probe, the above-described genomic library was screenecl again using the Tsp-l as a probe, which was labeled by the random priming.
Hybridization was carried out in a solution containing 5 x SSC, 1 x Denhardt's solution, 10~ dextran sulfate, 20 mM sodium phosphate, 400 ~ug,/ml human placental DNA and 98-- 3-1 6: 1 9: 33 i TAN I GAWA PATENT Sma r t 3 : ~332389 1 83 # 1 2/ ~ ~
the Tsp-l probe at 42~C for 18 hours. After the hybridization, the membranes were washed finally in O.l x SSC - 0.1% SDS at 52~C for 0.5 hours. The membranes were autoradiographed for 24 hours to Kodak Bio Max MS films at -70~C using an MS intensif-ying screen. As a result, a genomic clone, ~sp-2, originated from a normal allele was isolated.
A restriction map of Tsp-2 is shown in Fig. 3. The CAG repeat (open box) is located in the putative first exon (solid box).
Nucleotide sequence of the SmaI-ApaI fragment (630 bp) of Tsp-2 was determined. The sequence is shown in Fig. 4 and in SEQ ID NO:3 in the SEQUENCE LISTING. In Fig. 4, the region of which sequence was determined from both the genomic DNA and cDNA (described below) is shown in uppercase letters and the regions of which sequences were determined from the cDNA is shown in lowercase letters. The sites at which the base is different in the genomic D~A and the cDNA are shown in two ~ayers and the upper character indicate the base determined from the cDNA. The deduced amino acid sequence is shown below the nucleotide sequence. Translation initiated from the ATG
codon in the longest open reading frame in the putative first exon.
A human cerebral cortex cDNA library (Clonetech, Palo Alto, CA, USA~ was screened using oligonucleotides, F-l a~d R-l, designed to flank the CAG repeat as shown in 98- 3-16:19:33 ;TANIGAWA PATENT Sma~t3 ;0332389183 # 13,' 26 ~ig. 4, as the probes. Hybridization was performed for 18 hours at 55~C in a solution containing 6 x SSC, 10 x Denhardt's solution, 0.5% SDS, 0.05% sodium pyrophosphate, lOQ ng/ml sheared salmon sperm DNA and the end-labeled oligonucleotide probe. After the hybridization, the membranes were washed finally in 6 x SSC containing 0.5%
SDS and 0.05~ sodium pyrophosphate for 0.5 hours at 55~C.
A clone, FC-1, which gave a hybridization signal with both the oligonucleotide probes, was obtained. To identify the 5' end of the c~)NA, 5'-RACE was performed using 5'-RACE-Ready cDNA ~Clonetech, Palo Alto, CA, USA).
The primer R-1 was used in the primary PCR and the primer R-2 ~see Fig. 4) was used in the secondary ~CR. As the forward primer, F-1 (see Fig. 4) was used in both the primary and secondary PCR. The 5'-RACE product, 350 bp in size, was subcloned into a pT7Blue T vector (5 '~RACE-1). The identity of 5'-RACE--l was confirmed by the overlap of the nucleotide sequence with those of Fc-1, Tsp-l and Tsp-2. Nucleotide sequences were determined by the dideoxynucleotide chain termination method using double-stranded plasmid ~NAs as the templates.
PCR products obtained from genomic DNA of normal and SCA2-affected individuals using the primer pair of F-1 (forward side) and R-1 (reverse side) were cloned i~to a plasmid vector, pT7Blue, followed by determination of the nucleotide sequence. The results are shown in Fig. 5.
As shown in Fig. 5, the norma~ genes have one or two 9 8-- 3--1 6: 1 9: 3 3 ; TAN I GAWA P AT E N T CA 022323 1 1 1998 - 03 - 17 ; o 3 3 2 3 8 ~3 1 8 .: # 1 4 / 2 6 CAA, while SCA2 genes do not. Further/ in SCA2 genes, CCG CCG CAG may be inserted into the site immediately after the CAG repeat region.
Example 2 Measurement of CAG Repeat ~nits in Sample Numbers of CAG repeat units were determined by polyacrylamide gel electrophoresis analysis of PCR
products obtained using the primer pair cf F-1 and R-1.
PCR was performed in a total volume of 10 ~l containing 10 mM Tris-HCl, pH 8.3, 50 ~ KCl, 2.0 mM MgCl2, 1.7 M
N,N,N-trimethylgly~ine, lllK3q of [Q-32P]dCTP (111 Tbq/mmol), 30 ~M dCTP, and 200 ,uM each of dATP, dGTP and TTP, 0.25 ~M each of the two primers, 200 ng of genomic DNA and 1.25 U of Taq DNA polymerase. After an initial 2-min denaturation at 95~C, T'CR was performed for 32 cycles of 1-min denaturation at 95~C, 1-min annealing at 60~C and 1-min extension a~ 72~C, followed by a final extension at 72~C for 5 min. Sequence ladders obtained using the cloned genomic segments of the SCA2 gene, which contain various sizes of CAG repeats, were used as size markers. For normal alleles containing one or two CAA
interruptions, the numbers of the CAA units were included in the CAG repeat size. For SCA2 alleles having expanded CAG region, the above-menticned insert sequence immediately after the CAG region was not included in the size of the CAG region.
By the above-described method, the numbers of the CAG repeat units of normal individuals (286 chromosomes) 98- 3-16;19:33 ;TANIGAWA PATENTcA 02232311 1998-03-17 : n3323~918.~ # 15/ 2 and 10 pedigrees of SCA2 patients (34 SCA2 chromosomes) were determined. The results are shown in Fig. ~. In Fig. 6, open bars indicate the results of the normal genes and solid bars indicate the results of the SCA2 genes.
As is apparent from Fig.. 6, in all of the normal genes, the numbers of the CAG repeat units were not more than 24, while in all of the SCA2 genes, they were not less than 35. Thus, it was proved that genetic diagnosis of SCA2 can be attained by the method of the present invention.
The relationship betwee:n the number of the CAG
repeat units determined by the method described above and the age at onset of symptoms of SCA2 is shown in Fig. 7.
An inverse correlation was observed statistically. This suggests that expansion of the number of the CAG repeat units relates to the onset cf symptoms of SCA2.
INDUSTRIAL AVAILABILITY
By the present inventicn, genetic assay of SCA2 was first attained. Thus, it is expected that the present invention will greatly contribute ~o the diagnosis of SCA2.
98- 3--1 6; 1 9: 33 ; TAN I GAWA PATENT Sma r t 3 : 0332389 1 8~ # ' 6,' 2 5 SEQUENC~ LISTING
SEQ ID NO:1 SEQUENCE LENGTH: 355 SEQUENCE TYPE: nucleic acid STRNDEDNESS: double S~QUENCE DESCRIPTION
GGGACCGTAT CCCTCCGCCG CCCCTCCCCC GCCCGGCCCC GGCCCCCCTC CCTCCC~GCA 60 GAGCTCGGCT CCCTCCGCCT CAGACTGTTT TGGTA&CAAC GGCAACGGCG GCGGCGCGTT 120 Met Ser Leu Lys Pro SEQ ID NO:2 S~QUENCE LENGTH: 205 SEQUENCE TYPE: nucleic acid STRNDEDNESS: double SEQUENCE DESCRIPTION
CCG CC& CCC GCG GCT GCC AAT GTC CGC AAG CCC GGC GGC AGC GGC CTT 48 Pro Pro Pro Ala Ala Ala Asn Val Arg Lys Pro Gly Gly Ser Gly Leu Leu Ala Ser Pro Ala Ala Ala Pro Ser Pro Ser Ser Ser Ser Val Ser 98-- 3--1 6; 1 ~: 33 ;TAN I GAWA PATENT Sma r t3 : 03323891 8. # 1 7 ~ 2 5 CA 022323ll lsg8-03-l7 Ser Ser Ser Ala Thr Ala Pro Ser Ser Val Val Ala Ala Thr Ser Gly GGC GGG AGG CCC GGC CTG GGC AGGTGGGT~T CGGCACCCCA GCCCCCCTCC 19 Gly Gly Arg Pro Gly Leu Gly SEQ ID NO:3 SEQUENCE LENGTH: 623 SEQUENC~ TYPE: nucleic acid STRNDEDNESS: double SEQUENCE D~SCRI~TION
Met Ser Leu Lys Pro Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln lO 15 20 CA& CAG CAG CAG CAG CCG CCG CCC GCG GCT GCC M T GTC CGC AAG CCC 451 Gln Gln Gln Gln Gln Pro Pro Pro Ala Ala Ala Asn Val Arg Lys Pro 98- 3-1 6; 1 9: 33 ; TAN I GAWA PATENT Sma rt 3 ~ 03323891 8'-: # 1 8,/ 2~
Gly Gly Ser Gly Le~ Leu Ala Ser Pro Ala Ala Ala Pro Ser Pro Ser Ser S~r Ser Val Ser Ser Ser Ser Ala Thr Ala Pro Ser Ser Val Val ~5 60 65 GCG GCG ACC TCC GGC GGC GGG AGG CCC GGC CTG BGC AGGTG>GT ~93 Ala Ala Thr Ser Gly Gly Gly Arg Pro Gly Leu Gly SEQ ID N0:4 SEQUENCE LENGTH: 213 SEQUENCE TYPE: nucleic acid STRNDEDNESS: single SEQUENCE DESCRIPTION
CAGCAGCAGC A&CAGCA&CA GCAGCAGCAG CAGCAGCAGC AGCAGCAGCA GCAGCAGCAG 120 SEQ ID NO:5 SEQUENCE L~NGTH: 20 SEQUENCE TYPE: nucleic acid STRNDEDNESS: single SEQUENCE DESCRIPTION
98- 3-1 6: 1 9: 33 ;TAN I ~AWA PATENT Srra -t3 ; 03323891 83 # 1 9.' 26 SEQ ID NO:6 SEQUENCE LENGTH: 20 SEQ~ENCE TYPE: nucleic acid SEQUENC~ DESCRIPTION
SEQ ID NO:7 SEQUENCE LENGTH: 75 SEQUENCE TYPE: nuclelc acid SE~JENCE DESCRIPTION
SEQ ID NO:8 SEQUENCE LENGTH: 78 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION
CAGCAGCAGC AGCA&CAGCA GCAGCAACAG CAGC M CAGC AACAGCAGCA GCAGCAGCAG 60 SEQ ID NO:9 SEQUEN~E LENGTH: 78 SEQUENCE TYPE: nucleic acid SEQUENCE DESCRIPTION
CAGCAGCAGC AGCAGCAGCA GCAGCAGCAG C M CAGCA&C M CAGCAGCA GCAGCAGCAG 60 98- 3-1 6; 1 9: 33 ; TAN I GAWA PATENT Sma r t 3 ; 03323891 83 # 2 0,' 2 1~
SEQ ID NO:10 SEQUENCE LENGTH: 78 SEQUENCE TYP~: nucleic acid SEQUENCE DESCRIPTION
SEQ ID NO:11 SEQUENCE LE~GTH: 78 SEQUENCE TY~E: nucleic acid SEQUENCE DESCRIPTION
CAGCAGCAGC AGCAGCAGCA GC M CAGCAG CAGCAGCAGC AACAGCAGCA &CAGCAGCCG 60 SEQ ID NO:12 SEQUENCE LENGTH: 78 SEQUENCE TYPE: nucLeic acid SEQUENCE DESCRIPTION
Claims (7)
1. 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:2, and a test DNA as a template, and measuring the number of CAG
repeats in the amplified PCR product.
repeats in the amplified PCR product.
2. The method according to claim 1, wherein the number of the CAG repeat in the PCR product is measured by measuring the size of the PCR product.
3. The method according to claim 1, wherein the number of the CAG repeat is measured by sequencing the PCR
product.
product.
4. The method according to any one of claims 1 to 3, wherein the test DNA is a genomic DNA.
5. The method according to any one of claims 1 to 3, wherein the test DNA is a cDNA.
6. A primer which is a nucleic acid that hybridizes with a part of the nucleotide sequence shown in SEQ ID
NO:1 in the SEQUENCE LISTING, which is used in the method according to any one of claims 1 to 5.
NO:1 in the SEQUENCE LISTING, which is used in the method according to any one of claims 1 to 5.
7. A primer which is a nucleic acid that hybridizes with a part of the nucleotide sequence shown in SEQ ID
NO:2 in the SEQUENCE LISTING, which is used in the method according to any one of claims 1 to 5.
NO:2 in the SEQUENCE LISTING, which is used in the method according to any one of claims 1 to 5.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002232311A CA2232311A1 (en) | 1996-07-18 | 1996-07-18 | Method for diagnosis of spinocerebellar ataxia type 2 and primers therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002232311A CA2232311A1 (en) | 1996-07-18 | 1996-07-18 | Method for diagnosis of spinocerebellar ataxia type 2 and primers therefor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2232311A1 true CA2232311A1 (en) | 1998-01-29 |
Family
ID=4162227
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002232311A Abandoned CA2232311A1 (en) | 1996-07-18 | 1996-07-18 | Method for diagnosis of spinocerebellar ataxia type 2 and primers therefor |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2232311A1 (en) |
-
1996
- 1996-07-18 CA CA002232311A patent/CA2232311A1/en not_active Abandoned
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