Polymorphic analyses were performed for 57 Bulgarian CMT1 families. Patients were selected on the basis of genetic, clinical and electromyographic (EMG) data. All affected subjects underwent clinical and electrophysiological examinations by a team of neurologists to confirm CMT1 and exclude other types of peripheral neuropathy. The diagnosis was based on standard criteria of the European CMT Consortium (http://molgen-www.uia.ac.be/ CMT). All individuals gave their informed consent for genetic analysis after pre-testing and genetic counseling.

Blood samples were obtained from the individuals and DNA was isolated by a standard salting out procedure [8]. The PCR amplification of markers D17S122 and D17S921 (http://molgen-www.uia.ac.be/CMT) was performed using the following conditions, for marker D17S122: initial denaturation at 95°C/5 minutes, 26 cycles at 94°C/1 minute, 55°C/1 minute, 72°C/2 minutes, and final extension at 72°C/5 minutes. For marker D17S921: initial denaturation at 95°C/5 minutes, 26 cycles at 94°C/1 minute, 55°C/30 seconds, 72°C/30 seconds, and final extension at 72°C/5 minutes. The amplification mix contained a DNA template concentration between 20-200 ng/mL; 1 mM dNTP, 2 mM from each primer (the forward one was Cy5-labeled), 1x PCR buffer with 1.5 mM/2 mM MgCl₂ (for D17S122 and D17S921, respectively), 0.1 U/mL Taq DNA polymerase (STS Technologies, Sofia, Bulgaria). The PCR amplification was performed on a PCR apparatus (MJ Research Inc., Waltham, MA, USA).

The PCR products were separated on a 6% denaturing polyacrylamide gel on ALFexpressO (Pharmacia Biotech, Uppsala, Sweden). The area under the peak curves was determined by ALFexpressO/Fragment manager software. For statistical analysis of the data we used the WinStat4.3 program (StatSoft Inc., 1993).