Rapid real-time fluorescent PCR gene dosage test for the diagnosis of DNA duplications and deletions

Background: Current methods to determine gene dosage are time-consuming and labor-intensive. We describe a new and rapid method to assess gene copy nu mber for identification of DNA duplications or deletions occurring in Charc ot-Marie-Tooth disease type IA (CMT1A) and hereditary neuropathy with liabi lity to pressure palsies (HNPP), respectively. Methods: We studied-16 patients with HNPP, 4 with CMT1A, and 49 control sub jects. We used real-time PCR on the LightCycler system with use of a single capillary tube and no post-PCR handling A polymorphic fragment of the PMP2 2 gene was amplified to determine gene dosage for heterozygous samples. The presence of two alleles was used to indicate that no deletion was present in HNPP samples. The ratio obtained between the areas under each allele mel ting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitiv e gene dosage test,where the ratio between the areas under the melting curv es of the target DNA of samples and of the-competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. S amples from HNPP, CMT1A, and controls were analyzed. Results: Area ratios were similar to 0.6, 1.0, and 2.0 for HNPP, control,an d CMT1A samples, respectively. The results agreed with those obtained by So uthern blotting and microsatellite analysis in the same samples. Conclusions: Direct and competitive real-time fluorescent PCR can different iate one, two, or three copies of the-target DNA. The method described is s ensitive and accurate for detection of CMT1A duplications and HNPP deletion s and is faster and easier than current methods. (C) 2000 American Associat ion for Clinical Chemistry.