Titre : | Development of oligoarray methods for mutation detection and expression analysis of titin (TTN) and other muscle genes (abstract : congrès international de Myologie, 2005) |
contenu dans : | |
Auteurs : | Congrès international de myologie 2005 (International Congress of Myology 2005; 9-13 mai 2005; Nantes, France) ; Hackman P ; Pelin K ; Monni O ; Auvinen P ; Udd B |
Type de document : | Article |
Année de publication : | 2005 |
Pages : | p. 94 |
Langues: | Anglais |
Mots-clés : | colloque ; gène TTN ; génétique moléculaire ; mutation génétique ; myopathie distale de type Udd ; titine |
Résumé : |
Communication n° 468. Introduction : Tibial muscular dystrophy TMD/LGMD2J is caused by mutations in the TTN gene. Due to its huge size (363 exons encoding 38,138 amino acid residues), the search for new mutations can be a very laborious task using the conventional way of sequencing. A microarray-based mutation detection method would speed up the process of detecting mutations considerably. Objectives : To develop an oligoarray for detecting unknown mutations in TTN, and an expression oligoarray for analyzing the expression of different isoforms of titin and other muscular genes in different muscle tissues. Methods : Oligonucleotides of approximately 20 bp size, corresponding to wild type (Wt) TTN and nebulin (NEB), as well as known TTN mutations, were printed onto glass slides. The microarray were hybridized with mixtures of PCR products of Wt and mutated TTN and NEB, labelled with Cy5 (mutated DNA) and Cy3 (Wt). The unhybridized probes were washed using standard methods combined with an additional more stringent washing with TMAC (tetramethyl ammonium chloride). The results were analysed using IPLab DeArray image microarray software. In parallel with this, 50 mer oligonucleotide microarray will be constructed for the TTN gene as well as other sarcomeric genes to compare variation of gene expression. Results : The first hybridisations to the mutation chip showed clear differences between Wt and mutated samples. The clearest result was seen with the 11 nucleotide deletion/insertion FINmaj TTN mutation. The results for the smaller point mutations were less clear, but promising. Conclusions : The first results of the hybridisations indicates that this method might indeed provide new means as a way to detect even single base substitutions. At least larger mutations than single point mutations will be possible to screen for. The concept needs further development and refinement before it is ready to use and its efficacy is tested for the detection of previously unknown mutations. |