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Rapid detection of SMARCB1 sequence variation using high resolution melting

Dagar, Vinod, Chow, Chung-Wo, Ashley, David M. and Algar, Elizabeth M. 2009, Rapid detection of SMARCB1 sequence variation using high resolution melting, BMC cancer, vol. 9, no. article 437, pp. 1-9, doi: 10.1186/1471-2407-9-437.

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Title Rapid detection of SMARCB1 sequence variation using high resolution melting
Author(s) Dagar, Vinod
Chow, Chung-Wo
Ashley, David M.
Algar, Elizabeth M.
Journal name BMC cancer
Volume number 9
Issue number article 437
Start page 1
End page 9
Total pages 9
Publisher BioMed Central
Place of publication London, England
Publication date 2009
ISSN 1471-2407
Summary Background : Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM), for detecting sequence variations in SMARCB1. Methods : Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. Results : The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4%) showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA). A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. Conclusions : This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to facilitate whole gene mutation screening.
Language eng
DOI 10.1186/1471-2407-9-437
Field of Research 119999 Medical and Health Sciences not elsewhere classified
Socio Economic Objective 970111 Expanding Knowledge in the Medical and Health Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
Copyright notice ©2009, BioMed Central
Free to Read? Yes
Persistent URL http://hdl.handle.net/10536/DRO/DU:30052155

Document type: Journal Article
Collections: School of Medicine
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Every reasonable effort has been made to ensure that permission has been obtained for items included in DRO. If you believe that your rights have been infringed by this repository, please contact drosupport@deakin.edu.au.