Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 86

Abstract

At least 15 genetic loci and ten genes have been associated with the demyelinating form of Charcot‐Marie‐Tooth hereditary neuropathy and related disorders. As pathogenic mutations have been identified in more and more genes, the labor and expense of screening for such mutations has increased substantially. Using conventional methods such as direct sequencing, single‐strand conformation polymorphism (SSCP), or heteroduplex analysis, molecular diagnosis of CMT remains laborious due to the large number of exons to be screened. Some genes (MPZ, PMP22, EGR2, GJB1) are small and have to be analyzed in a relatively large number of patients while other rarer genes, such as periaxin (PRX, 19q13), are too large (7 exons encoding transcripts of 4.8–5.5 kilobases) to be easily included in a molecular diagnostic panel. Furthermore, the sensitivity of the more economical techniques (SSCP and heteroduplex analysis) is far from being optimal ( ). Denaturing High‐Performance Liquid Chromatography (DHPLC) is a newly developed method to scan DNA for mutations, which is capable of automated high‐throughput analysis that does not require modified PCR primers, specific reagent arrays or detection labels, or any sample pretreatment other than PCR. We have undertaken the mutational analysis of some genes associated with CMT1 and related neuropathies by using DHPLC. Optimal conditions have been determined for DHPLC analysis of MPZ, PMP22, GJB1 and PRX genes in an initial group of 30 patients with severe demyelinating neuropathy, negative for the common 17p11 duplication. The very high sensitivity of the method was demonstrated by analyzing a control group of patients with known mutations in the MPZ, PMP22 and GJB1 genes. The work is still in progress. Thus far, DHPLC has resolved all the mutations previously detected by sequencing and allowed the identification of a number of polymorphic variants in the PRX gene and a novel disease mutation in MPZ gene exon 4 in a patient with an intermediate (axonal‐demyelinating) form of CMT. Interestingly, this mutation had consistently appeared as homozygous at previous direct sequencing analysis. DHPLC clearly showed the heterozygous state of the mutation, thus demonstrating the power of this technique also in identifying sequencing artifact or mutation mosaicism, if any. Partially supported by a grant Ricerca Finalizzata Ministero della Sanità to FT.