New polymorphic short tandem repeats for PCR-based Charcot-Marie-Tooth disease type IA duplication diagnosis

Background: Charcot-Marie-Tooth disease type 1A (CMT1A) accounts for 70-90% of eases of CMT1 and is most frequently caused by the tandem duplication o f a 1.4-Mb genomic fragment on chromosome 17p12. Molecular diagnosis of CMT 1A has been based primarily on pulsed-field electrophoresis, fluorescence i n situ hybridization, polymorphic allele dosage analysis, and quantitative PCR, We sought to improve the fidelity and applicability of PCR-based diagn osis by developing a panel of novel, highly polymorphic short tandem repeat s (STRs) from within the CMT1A duplicated region. Methods: We used a recently available genomic sequence to identify potentia lly polymorphic simple repeats. We then amplified these sequences in a mult iethnic cohort of unaffected individuals and assessed the heterozygosity an d number of alleles for each STR. Highly informative markers were then test ed in a set of previously diagnosed CMT1A duplication patients, and the abi lity to identify the genomic duplication through the presence of three band s was assessed. Results: We identified 34 polymorphic markers, 15 of which were suitable fo r CMT1A diagnosis on the basis of high heterozygosity in different ethnic g roups, peak uniformity, and a large number of alleles. On the basis of the fluorescent dye and allele range of each marker, we developed two panels, e ach of which could be analyzed concurrently. Panel 1, which comprised 10 ma rkers, detected 37 of 39 duplications, whereas panel 2, which comprised the remaining 5 markers, identified 21 of 39 duplications, Through the combina tion of both panels, we identified 39 of 39 duplications in previously diag nosed CMT1A patients. Conclusions: The newly developed 15-marker set has the capability of detect ing > 99% of duplications and thus is a powerful and versatile diagnostic t ool, (C) 2001 American Association for Clinical Chemistry.