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H. Osada, H. Yaguchi, H. Sasaki, S. Liu, H. Yonekura; Identification of the Mutation Site of Stickler Syndrome Causative Gene in Rna Extracted From Peripheral Blood Leukocyte. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2813.
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Since type 1 Stickler syndrome patients have mutations in type 2 collagen gene(COL2A1) which is expressed specifically in eyes and cartilages, identification of the genetic mutation site is necessary in making diagnosis. However, COL2A1 is so long that it requires considerable labor to analyze by genome sequencing. Therefore, we isolated mRNA of leaking mutant gene extracted from peripheral blood leukocyte, and analyzed it to identify the mutation site. The record is reported herewith.
First, we performed a differential count and separated leukocytes from a blood sample of a patient(male, 18 yr old) suspected to have Stickler syndrome, and cultivated them under the presence of cycloheximide for 4 h to extract RNA. Next, COL2A1 cDNA was amplified and isolated by RT-PCR, and the mutation was identified by base sequence determination. Then genomic DNA was isolated from blood samples of this patient and 3 family members(41 yr old father, 18 and 15 yr old brothers) also suspected of having Stickler syndrome, and mutation sites were examined.
49 base pair deletion was evident in the translated region of mutated mRNA. The deletion induced twists of subsequent translation domain resulting in major change of amino-acid sequence. By sequencing and analysis of the genome region corresponding to the deleted part, a single base mutation in the 3’splice site of intron17 was noted, and considered to be the cause of abnormal splicing. The same mutants were found in all 4 subjects.
We succeeded in identification of the mutation site in a Stickler syndrome patient by isolating and analyzing mRNA of the mutant gene extracted from peripheral blood leukocyte. This technique is simple and expected to be useful in diagnosis of other hereditary ophthalmic disease.
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