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Biochem Biophys Res Commun. 2011 Feb 18;405(3):36872. doi: 10.1016/j.bbrc.2011.01.011. Epub 2011 Jan 8.
A novel mutation screening system for EhlersDanlos Syndrome,
vascular type by highresolution melting curve analysis in
combination with small amplicon genotyping using genomic DNA.
Naing BT1, Watanabe A, Shimada T.
Author information
Abstract
EhlersDanlos syndrome, vascular type (vEDS) (MIM #130050) is an autosomal dominant
disorder caused by type III procollagen gene (COL3A1) mutations. Most COL3A1 mutations are
detected by using total RNA from patientderived fibroblasts, which requires an invasive skin
biopsy. Highresolution melting curve analysis (hrMCA) has recently been developed as a post
PCR mutation scanning method which enables simple, rapid, costeffective, and highly sensitive
mutation screening of large genes. We established a hrMCA method to screen for COL3A1
mutations using genomic DNA. PCR primers pairs for COL3A1 (52 amplicons) were designed to
cover all coding regions of the 52 exons, including the splicing sites. We used 15 DNA samples (8
validation samples and 7 samples of clinically suspected vEDS patients) in this study. The eight
known COL3A1 mutations in validation samples were all successfully detected by the hrMCA. In
addition, we identified five novel COL3A1 mutations, including one deletion (c.2187delA) and one
nonsense mutation (c.2992C>T) that could not be determined by the conventional total RNA
method. Furthermore, we established a small amplicon genotyping (SAG) method for detecting
three high frequency codingregion SNPs (rs1800255:G>A, rs1801184:T>C, and rs2271683:A>G)
in COL3A1 to differentiate mutations before sequencing. The use of hrMCA in combination with
SAG from genomic DNA enables rapid detection of COL3A1 mutations with high efficiency and
specificity. A better understanding of the genotypephenotype correlation in COL3A1 using this
method will lead to improve in diagnosis and treatment.
Copyright © 2011 Elsevier Inc. All rights reserved.
PMID: 21219851 DOI: 10.1016/j.bbrc.2011.01.011
[Indexed for MEDLINE]
Publication types, MeSH terms, Substances
Publication types
Evaluation Studies
Research Support, NonU.S. Gov't
https://www.ncbi.nlm.nih.gov/pubmed/21219851 1/2
8/8/2017 A novel mutation screening system for Ehlers-Danlos Syndrome, vascular type by high-resolution melting curve analysis in combination with small amp... …
MeSH terms
Collagen Type III/genetics*
DNA Primers/genetics
EhlersDanlos Syndrome/diagnosis*
EhlersDanlos Syndrome/genetics
Genetic Testing/methods*
Genotype
Humans
Mutation
Polymerase Chain Reaction/methods*
Polymorphism, Single Nucleotide
Transition Temperature
Substances
COL3A1 protein, human
Collagen Type III
DNA Primers
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https://www.ncbi.nlm.nih.gov/pubmed/21219851 2/2