Modifikation der enzymatischen Mutationsdetektionsmethode (EMD) zur Identifikation von Punktmutationen im PMP22-Gen nach DNA-Isolierung aus Nerv, Muskel oder Blut
Hereditary Motor and Sensory Neuropathies (HMSN) are a group of genetically determined disorders of the peripheral nervous system, which are clinically characterised by chronic, progressive weakness of the lower limbs, muscular atrophy and sensibility disorders. Phenotypic variability, however, is quite great, which makes it often difficult to identify the exact HMSN subtype. Therefore and for a better understanding of the underlying pathological mechanisms, molecular genetic analysis is very important. In this study, DNA form 59 patients was examined for point mutations in the PMP22-Gene, which codes for a transmembrane protein in the myelin sheath. After DNA-extraction, the four coding exons of the gene were amplified by PCR. Then, the templates were analysed by the Enzymatic Mutation Detection Method (EMD): In case of a heterozygous point mutation, hybridisation of two amplified DNA strands causes a base pair mismatch, which is detected by the T4 Endonuclease VII. The enzyme cleaves the DNA duplex at that point. For the first time, the fragments were analysed on the ABI Prism 310. Based on capillary electrophoresis, this system separates and detects fluorescence labelled DNA-fragments. To adapt EMD for the ABI Prism and optimise the results, several steps had been necessary. A presumed base exchange was identified exactly by Automatic Sequencing Analysis. In 58 patients, a mutation in the PMP22-gene was excluded. In one patient, a heterozygous point mutation was identified in exon 3 (codon 97, exchange of the third base Cytosin with Thymin). As this exchange does not alter the amino acid sequence, this mutation is assumed to be a polymorphism without pathological effect. To summarize, these results show how rare sequence variations in the PMP22-gene are and form the base for further investigations (e.g. the analysis of other with HMSN associated genes as P0, EGR2, CX32 etc.). Moreover, adapting and optimizing EMD for the ABI Prism 310 allowed the use of a very modern, efficient and reliable screening method with a sensitivity of nearly 100%. For the first time, this method has been used in combination with Sequencing Analysis to search for point mutations in HMSN Patients.
info:eu-repo/classification/ddc/610, Medizin, HMSN, EMD, PMP22, CMT1, HNPP, CH, DSS
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