Detection of genomic rearrangements by DHPLC: a prospective study of 90 patients with inherited peripheral neuropathies associated with 17p11.2 rearrangements.

Large genomic duplications and deletions are increasingly recognized as a cause of human disease. Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsy (HNPP) result, respectively, from a duplication or deletion of a 1.5 Mb genomic region in 17p11.2-12, containing the PMP22 gene. In routine diagnostic analysis, CMT1A status is inferred from the detection of an imbalanced dosage of two alleles or the presence of three alleles of a polymorphic marker flanking the PMP22 gene. HNPP is suspected if only one allele is seen, but hemizygosity must be confirmed by analyzing allele segregation in the family or by other techniques such as Southern blotting or fluorescence in situ hybridization (FISH). PCR-based methodologies have also been developed that allow single-step determination of the PMP22 gene copy number, wherein amplicons are typically labeled and/or separated by gel electrophoresis. We describe here a fast and reliable PCR-based method for the diagnosis of CMT1A and HNPP in which the PMP22 gene is co-amplified with a reference gene, and the amplicons are separated according to their size and quantified by DHPLC. Our results suggest that this method for quantifying gene dosage could be applied to other genomic rearrangements.