| Project/Area Number |
18591926
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Ophthalmology
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| Research Institution | Fukuoka University (2007)
Kyushu University (2006) |
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Principal Investigator |
YOSHIDA Shigeo Fukuoka University, Chikushi Hospital, Senior Assistant Professor (50363370)
|
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| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Ken Kyusyu University, Molecular Genetics, Associate Professor (60274528)
YOSHIMASU Koichi Wakayama Medical University, Facutly of Medicine, Senior Assistant Professor (40382337)
OZOEGAWA Manabu Kyushu University, Neurology, Assistant Professor (60380614)
ISHIBASHI Tatsuro Kyusyu University, Ophthalmology, Professor (30150428)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,980,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Genetics / Rapid diagnosis / Molecular diagnosis / DNAチップ / 遺伝性眼疾患 |
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| Research Abstract |
1. To report a novel mutation in the GUCY2D gene in a Japanese family with autosomal dominant cone-rod dystrophy (adCORD), and to examine the possible use of arrayed primer extension (APEX)-based genotyping chip in detecting mutations, genomic DNA was extracted from the peripheral blood of family members. It was PCR-amplified, fragmented, and hybridized to APEX-based genotyping microarrays on which known disease-associated sequence variations were arrayed for patients with early-onset retinal dystrophy. Five single nucleotide polymorphisms in AIPLI, RPGRIPI, and GUCY2D were detected in the proband by microarray screening, and all were validated by direct sequencing. A novel heterozygous triple missense mutation of c.2540_2542delinsTCC (p.G1n847_Lys848delinsLeuGln amino acid substitutions) was found in both the proband and his affected father, and the three nucleotide changes were located on the same chromosome. Our study demonstrates that the APEX-based gene screening can be used to id
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entify simultaneously disease-modifying sequence changes as well as disease-causing mutations.
2. Mutation 926delA of the arrestin/S-antigen SAG gene is the main cause of Oguchi disease in the Japanese. The purpose of this study was to develop a rapid diagnostic assay to detect mutations in the SAG gene. Two sequence-specific primers and fluorophore-labeled probes for exon 11 of the SAG gene were designed, and the region spanning the mutations was amplified by polymerase chain reaction (PCR) using the LightCycler detection system. The mutations were then identified by melting curve analyses of the hybrid formed between the PCR product and a specific fluorescent probe.
We clearly distinguished each SAG genotype (homozygous and heterozygous 926delA and wild type) by the distinct melting peaks at different temperatures. One thermal cycling required approximately 54 min to process, and the results were 100% in concordance with the genotypes determined by DNA sequencing. We have succeeded in developing a rapid method to detect the most frequent mutation in the SAG gene. Less
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