Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses

Carol Dobson-Stone, Roger D. Cox, Lorne Lonie, Lorraine Southam, Maria Fraser, Carol Wise, François Bernier, Shirley Hodgson, Daniel E. Porter, A. Hamish R W Simpson, Anthony P. Monaco

Research output: Contribution to journalArticle

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Abstract

EXT1 and EXT2 are two genes responsible for the majority of cases of hereditary multiple exostoses (HME), a dominantly inherited bone disorder. In order to develop an efficient screening strategy for mutations in these genes, we performed two independent blind screens of EXT1 and EXT2 in 34 unrelated patients with HME, using denaturing high-performance liquid chromatography (DHPLC) and fluorescent single-strand conformation polymorphism analysis (F-SSCP). The mutation likely to cause HME was found in 29 (85%) of the 34 probands: in 22 of these (76%), the mutation was in EXT1; seven patients (24%) had EXT2 mutations. Nineteen of these disease mutations have not been previously reported. Of the 42 different amplicon variants identified in total in the cohort, 40 were detected by DHPLC and 39 by F-SSCP. This corresponds to mutation detection efficiencies of 95% and 93% respectively. We have also found that we can confidently distinguish between different sequence variants in the same fragment using F-SSCP but not DHPLC. In light of this, and the similarly high sensitivities of the two techniques, we propose to continue screening with F-SSCP.

Original languageEnglish (US)
Pages (from-to)24-32
Number of pages9
JournalEuropean Journal of Human Genetics
Volume8
Issue number1
DOIs
StatePublished - Jan 2000

Fingerprint

Multiple Hereditary Exostoses
High Pressure Liquid Chromatography
Mutation
Genes
Bone and Bones

Keywords

  • Denaturing high-performance liquid chromatography
  • Diaphyseal aclasis
  • EXT1
  • EXT2
  • Hereditary multiple exostoses
  • Mutation detection
  • Single-strand conformation polymorphism analysis
  • SNP detection

ASJC Scopus subject areas

  • Genetics(clinical)

Cite this

Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses. / Dobson-Stone, Carol; Cox, Roger D.; Lonie, Lorne; Southam, Lorraine; Fraser, Maria; Wise, Carol; Bernier, François; Hodgson, Shirley; Porter, Daniel E.; Simpson, A. Hamish R W; Monaco, Anthony P.

In: European Journal of Human Genetics, Vol. 8, No. 1, 01.2000, p. 24-32.

Research output: Contribution to journalArticle

Dobson-Stone, Carol ; Cox, Roger D. ; Lonie, Lorne ; Southam, Lorraine ; Fraser, Maria ; Wise, Carol ; Bernier, François ; Hodgson, Shirley ; Porter, Daniel E. ; Simpson, A. Hamish R W ; Monaco, Anthony P. / Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses. In: European Journal of Human Genetics. 2000 ; Vol. 8, No. 1. pp. 24-32.
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abstract = "EXT1 and EXT2 are two genes responsible for the majority of cases of hereditary multiple exostoses (HME), a dominantly inherited bone disorder. In order to develop an efficient screening strategy for mutations in these genes, we performed two independent blind screens of EXT1 and EXT2 in 34 unrelated patients with HME, using denaturing high-performance liquid chromatography (DHPLC) and fluorescent single-strand conformation polymorphism analysis (F-SSCP). The mutation likely to cause HME was found in 29 (85{\%}) of the 34 probands: in 22 of these (76{\%}), the mutation was in EXT1; seven patients (24{\%}) had EXT2 mutations. Nineteen of these disease mutations have not been previously reported. Of the 42 different amplicon variants identified in total in the cohort, 40 were detected by DHPLC and 39 by F-SSCP. This corresponds to mutation detection efficiencies of 95{\%} and 93{\%} respectively. We have also found that we can confidently distinguish between different sequence variants in the same fragment using F-SSCP but not DHPLC. In light of this, and the similarly high sensitivities of the two techniques, we propose to continue screening with F-SSCP.",
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