title: Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by "dual-fluorescence labeled PCR-restriction fragment length analysis".
abstract: Spinocerebellar ataxia type 1 (SCA1; OMIM: #164400) is an autosomal dominant cerebellar ataxia caused by an expansion of CAG repeat, which encodes polyglutamine, in the ataxin-1 (ATXN1) gene. Length of polyglutamine in the ATXN1 protein is the critical determinant of pathogenesis of this disease. Molecular diagnosis of SCA1 is usually undertaken by assessing the length of CAG repeat configuration using primers spanning this configuration. However, this conventional method may potentially lead to misdiagnosis in assessing polyglutamine-encoding CAG repeat length, since CAT interruptions may be present within the CAG repeat configuration, not only in normal controls but also in neurologically symptomatic subjects. We developed a new method for assessing actual CAG repeat numbers not interrupted by CAT sequences. Polymerase chain reaction using a primer pair labeled with two different fluorescences followed by restriction enzyme digestion with SfaNI which recognizes the sequence "GCATC(N)(5)", lengths of actual CAG repeats that encode polyglutamine were directly detected. We named this method "dual fluorescence labeled PCR-restriction fragment length analysis". We found that numbers of actual CAG repeat encoding polyglutamine do not overlap between our cohorts of normal chromosomes (n=385) and SCA1 chromosomes (n=5). We conclude that the present method is a useful way for molecular diagnosis of SCA1.
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Jiang X
authors.initials: JX
authors.lastname: Lin
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan. kishikawa.nuro@tmd.ac.jp.
authors.forename: Kinya
authors.initials: K
authors.lastname: Ishikawa
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Sakamoto
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Taiji
authors.initials: T
authors.lastname: Tsunemi
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Ishiguro
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Amino
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Shuta
authors.initials: S
authors.lastname: Toru
authors.affiliation: Department of Medical Genetics, Ehime University, Onsen-gun, Ehime, Japan.|Ibaraki Prefectural Child Welfare Health Center, Yoshizawa-cho 3979-3, Mito, Ibaraki, 310-0845, Japan.
authors.forename: Ikuko
authors.initials: I
authors.lastname: Kondo
authors.affiliation: Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Mizusawa
mesh_terms: D000067696:Ataxin-1; D000067528:Ataxins; D017931:DNA Primers / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D020754:Spinocerebellar Ataxias / Q000235:genetics*; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
chemical_list: C000593749:ATXN1 protein, human; D000067696:Ataxin-1; D000067528:Ataxins; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins