Abstract：Objective:To establish high-resolution melting analysis(HRMA)assay and detect the SF3B1 mutations in patients with myelodysplastic syndrome(MDS). Methods: The PCR amplification systems with internal temperature calibrators were established with specific PCR primers against four hotspot mutation sites of SF3B1 gene(E622, H662, K666, and K700). Thirty MDS samples were amplified and analyzed by using HRMA technique. The results of HRMA were verified by DNA sequencing. Results: Abnormal melting curves were observed by HRMA assay in two MDS samples. DNA sequencing confirmed that these two samples harbored heterozygous K666M mutation and K700E mutation, respectively. The sensitivity of HRMA assay could reach 0.05. Conclusion: The HRMA assay for detecting SF3B1 mutations was established successfully and could be used to screen SF3B1 mutations rapidly in patients with MDS.
杨静1, 姚冬明1,钱军1,王翠竹1,杨小飞1,林江2,李云1, 陈星星1,肖高飞2,马吉春2. 建立高分辨熔解曲线分析法检测SF3B1基因突变[J]. 江苏大学学报:医学版, 2013, 23(1): 49-52.
YANG Jing1, YAO Dong-ming1, QIAN Jun1, WANG Cui-zhu1,YANG Xiao-fei1, LIN Jiang2, LI Yun1, CHEN Xing-xing1,XIAO Gao-fei2. Detection of SF3B1 mutations using high-resolution melting analysis. Journal of Jiangsu University(Medicine Edition), 2013, 23(1): 49-52.
［1］ Pajares MJ, Ezponda T, Catena R, et al. Alternative splicing: an emerging topic in molecular and clinical oncology［J］. Lancet Oncol, 2007, 8(4):349-357.［2］ David CJ, Manley JL. Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged［J］. Genes Dev, 2010, 24(21):2343-2364.［3］ Grosso AR, Martins S, Carmo Fonseca M. The emerging role of splicing factors in cancer［J］. EMBO Rep, 2008, 9(11):1087-1093.［4］ Golas MM, Sander B, Will CL, et al. Molecular architecture of the multiprotein splicing factor SF3b［J］. Science, 2003, 300(5621):980-984.［5］ Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia［J］. Nature, 2011, 478(7367):64-69.［6］ Papaemmanuil E, Cazzola M, Boultwood J, et al. Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts［J］. N Engl J Med, 2011, 365(15):1384-1395.［7］ Malcovati L, Papaemmanuil E, Bowen DT, et al. Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic／myeloproliferative neoplasms［J］. Blood, 2011, 118(24):6239-6246.［8］ Damm F, Thol F, Kosmider O, et al. SF3B1 mutations in myelodysplastic syndromes: clinical associations and prognostic implications［J］. Leukemia, 2012, 26(5):1137-1140.［9］ Patnaik MM, Lasho TL, Hodnefield JM, et al. SF3B1 mutations are prevalent in myelodysplastic syndromes with ring sideroblasts but do not hold independent prognostic value［J］. Blood, 2012, 119(2):569-572.［10］ Wang L, Lawrence MS, Wan Y, et al. SF3B1 and other novel cancer genes in chronic lymphocytic leukemia［J］. N Engl J Med, 2011, 365(26):2497-2506.［11］ Rossi D, Bruscaggin A, Spina V, et al. Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia: association with progression and fludarabine-refractoriness［J］. Blood, 2011, 118(26):6904-6908.［12］ Montgomery J, Wittwer CT, Palais R, et al. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis［J］. Nat Protoc, 2007, 2(1):59-66.［13］ Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization(WHO)classification of myeloid neoplasms and acute leukemia: rationale and important changes［J］. Blood, 2009, 114(5):937-951.［14］ Qian J, Lin J, Yao DM, et al. Rapid detection of JAK2 V617F mutation using high-resolution melting analysis with LightScanner platform［J］. Clin Chim Acta, 2010, 411(23/24): 2097-2100.［15］ Nagai K, Muto Y, Pomeranz Krummel DA, et al. Structure and assembly of the spliceosomal snRNPs. Novartis Medal Lecture［J］. Biochem Soc Trans, 2001, 29(Pt 2): 15-26.
2013, Vol. 23 
