Background: A 1.5-Mb microduplication containing the gene for peripheral myelin protein 22 (PMP22) on chromosome 17p11.2-12 is responsible for 75% of cases of the demyelinating form of Charcot-Marie-Tooth disease (CMT1A). Methods for molecular diagnosis of CMT1A use Southern blot and/or amplification by PCR of polymorphic poly(AC) repeats (microsatellites) located within the duplicated region, or the detection of junction fragments specific for the duplication. Difficulties with both strategies have led us to develop a new diagnostic strategy with highly polymorphic short tandem repeats (STRs) located inside the CMT1A duplicated region.
Methods: We tested 10 STRs located within the duplication for polymorphic behavior. Three STRs were selected and used to test a set of 130 unrelated CMT1A patients and were compared with nonduplicated controls. The study was then extended to a larger population of patients. Alleles of interest were sequenced. A manual protocol using polyacrylamide electrophoresis and silver staining and an automated capillary electrophoresis protocol to separate fluorescently labeled alleles were validated.
Results: We identified three new STRs covering 0.55 Mb in the center of the CMT1A duplication. One marker, 4A, is located inside the PMP22 gene. The two others, 9A and 9B, more telomerically positioned, have the highest observed heterozygosity reported to date for CMT1A markers: 0.80 for 9A, and 0.79 for 9B. Tetra- and pentanucleotide repeats offered clear amplification, accurate sizing, and easy quantification of intensities.
Conclusions: Combined use of the three STRs allows robust diagnosis with almost complete informativeness. In our routine diagnosis for CMT1A, they have replaced the use of other polymorphic markers, either in a manual adaptation or combined with fluorescence labeling and allele sizing on a DNA sequencer.