Abstract:
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Mutation detection in nemaline myopathy (NM) is challenging due to the size of the main causative gene, nebulin (NEB), with 183 exons. Using denaturing high performance liquid chromatography (dHPLC) we have identified 91 different exonic and intronic point mutations as well as small deletions and insertions in NEB in 85 families. Carefully optimised dHPLC offers efficient identification of heterozygous mutations. The identification of the first big deletion in NEB erasing the entire exon 55 (Anderson, 2004) revealed the importance of a convenient method for tracking larger alterations (large deletions, insertions or amplifications) affecting whole exons of NEB. Multiplex Ligation-Dependent Probe Amplification (MLPA) is a promising method for the detection of DNA copy number changes. In MLPA, a copy is made of each target exon by hybridization of two probes to each exon. The probes are ligated and then amplified in a multiplex-PCR reaction using universal fluorescent-labelled primer pair. The PCR fragments produced are analysed by fragment analysis methods, and the relative copy numbers of the target exons are calculated. Our ongoing MLPA studies currently include synthetic self-designed probes for 30% of NEB exons. The initial series consists of DNA samples from nine NM patients with one known NEB mutation each. Altogether 167 different probe pairs are required for analysis of all NEB exons, as the same eight probe pairs will hybridise to the highly homologous exons 82-105 in the central region of NEB. In addition to these ongoing mutation analyses we screen the families for the deletion of exon 55 using Anderson’s PCR-based method. Where myoblast RNA is available we perform RT-PCR in order to sequence the constitutively spliced regions of NEB mRNA. RT-PCR has revealed three novel mutations in NEB. Efficient mutation identification in NEB requires combination of multiple methods.
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