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Rapid detection of SMARCB1 sequence variation using high resolution melting
Vinod Dagar1, Chung-Wo Chow, David M Ashley
1Children's Cancer Centre, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia. vinod.dagar@mcri.edu.au
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.
Insights
High-resolution melting (HRM) analysis offers a rapid, sensitive, and cost-effective method for screening SMARCB1 gene mutations in rhabdoid tumors. This approach aids in assessing cancer recurrence and transmission risks.
Area of Science:
- Oncology
- Genetics
- Molecular Diagnostics
Background:
- Rhabdoid tumors are aggressive pediatric cancers linked to SMARCB1 gene mutations.
- Germline SMARCB1 inactivation is associated with rhabdoid tumors, epithelioid sarcoma, and familial schwannomatosis.
- Accurate mutation screening is crucial for risk assessment in affected families.
Purpose of the Study:
- To develop and validate a rapid, sensitive diagnostic screening method for SMARCB1 sequence variations.
- To evaluate the utility of high-resolution melting (HRM) analysis for mutation detection in SMARCB1.
Main Methods:
- Screening of SMARCB1 coding exons, splice sites, and UTRs using HRM and direct DNA sequencing.
- Optimization of HRM reaction conditions with commercial reagents.
- Confirmation of mutations using digital HRM and re-sequencing for complex cases.
Main Results:
- HRM demonstrated zero false negatives in the tested sample series.
- Nine distinct pathogenic SMARCB1 mutations were identified in 19 rhabdoid tumors.
- Detection sensitivity varied based on sequence context and nucleotide change.
Conclusions:
- HRM is a reliable and efficient tool for SMARCB1 mutation screening.
- This method is suitable for widespread adoption in diagnostic laboratories.
- HRM facilitates comprehensive gene mutation screening for rhabdoid tumors.
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