Implications for CMT 1X Gene Mutation Scanning: GJB1 with High-Resolution Melting A

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Clin Chem. 2007 Jan 2

Mutation Scanning the GJB1 Gene with High-Resolution Melting

Analysis: Implications for Mutation Scanning of Genes for Charcot-

Marie-Tooth Disease.

Kennerson ML, Warburton T, Nelis E, Brewer M, Polly P, De Jonghe P,

Timmerman V, Nicholson GA.

Northcott Neuroscience Laboratory, ANZAC Research Institute, Concord

NSW, Australia 2139, and Molecular Medicine Laboratory, Concord

Hospital, Concord NSW, Australia.

BACKGROUND: X-linked Charcot-Marie-Tooth type 1 disease has been

associated with 280 mutations in the GJB1 [gap junction protein,

beta 1, 32kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-

linked)] gene. High-resolution melting analysis with an automated

instrument can be used to scan DNA for alterations, but its use in X-

linked disorders has not been described.

METHODS: A 96-well LightScanner for high resolution melting analysis

was used to scan amplicons of the GJB1 gene. All mutations reported

in this study had been confirmed previously by sequence analysis.

DNA samples were amplified with the double-stranded DNA-binding dye

LC Green Plus. Melting curves were analyzed as fluorescence

difference plots. The shift and curve shapes of melting profiles

were used to distinguish controls from patient samples.

RESULTS: The method detected each of the 23 mutations used in this

study. Eighteen known mutations provided validation of the high-

resolution melting method and a further 5 mutations were identified

in a blind study. Altered fluorescence difference curves for all the

mutations were easily distinguished from the wild-type melting

profile.

CONCLUSION: High-resolution melting analysis is a simple, sensitive,

and cost-efficient alternative method to scan for gene mutations in

the GJB1 gene. The technology has the potential to reduce sequencing

burden and would be suitable for mutation screening of exons of

large multiexon genes that have been discovered to be associated

with Charcot Marie Tooth neuropathy.