Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by "dual-fluorescence labeled PCR-restriction fragment length analysis".
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.
Lin
Jiang X
JX
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
Ishikawa
Kinya
K
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.
Sakamoto
Masaki
M
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
Tsunemi
Taiji
T
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
Ishiguro
Taro
T
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
Amino
Takeshi
T
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
Toru
Shuta
S
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
Kondo
Ikuko
I
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.
Mizusawa
Hidehiro
H
Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
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*
C000593749:ATXN1 protein, human; D000067696:Ataxin-1; D000067528:Ataxins; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins