Polymorphic short tandem repeats for diagnosis of the Charcot-Marie-Tooth IA duplication

Background: A 1.5-Mb microduplication containing the gene for peripheral my elin protein 22 (PMP22) on chromosome 17p11.2-12 is responsible for 75% of cases of the demyelinating form of Charcot-Marie-Tooth disease (CMT1A). Met hods for molecular diagnosis of CMT1A use Southern blot and/or amplificatio n by PCR of polymorphic poly(ae) repeats (microsatellites) located within t he duplicated region, or the detection of junction fragments specific for t he 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 b ehavior. Three STRs were selected and used to test a set of 130 unrelated C MT1A 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 silve r 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 t wo others, 9A and 9B, more telomerically positioned, have the highest obser ved heterozygosity reported to Bate for CMT1A markers: 0.80 for 9A, and 0.7 9 for 9B. Tetra- and pentanucleotide repeats offered clear amplification, a ccurate sizing, and easy quantification of intensities. Conclusions: Combined use of the three STRs allows robust diagnosis with al most complete informativeness. In our routine diagnosis for CMT1A, they hav e replaced the use of other polymorphic markers, either in a manual adaptat ion or combined with fluorescence labeling and allele sizing on a DNA seque ncer. (C) 2001 American Association for Clinical Chemistry.