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 number for identification of DNA duplications or deletions occurring in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively.


METHODS
We studied 16 patients with HNPP, 4 with CMT1A, and 49 control subjects. 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 PMP22 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 melting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitive gene dosage test, where the ratio between the areas under the melting curves of the target DNA of samples and of the competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. Samples from HNPP, CMT1A, and controls were analyzed.


RESULTS
Area ratios were approximately 0.6, 1.0, and 2.0 for HNPP, control, and CMT1A samples, respectively. The results agreed with those obtained by Southern blotting and microsatellite analysis in the same samples.


CONCLUSIONS
Direct and competitive real-time fluorescent PCR can differentiate one, two, or three copies of the target DNA. The method described is sensitive and accurate for detection of CMT1A duplications and HNPP deletions and is faster and easier than current methods.
Ruiz-Ponte
C
C
Molecular Medicine Unit-INGO (Sergas), University of Santiago de Compostela, Hospital de Conxo, 15706 Santiago de Compostela, Spain.
Loidi
L
L
Vega
A
A
Carracedo
A
A
Barros
F
F
D002607:Charcot-Marie-Tooth Disease / Q000235:genetics; D004247:DNA / Q000235:genetics*; D005453:Fluorescence; D017353:Gene Deletion; D018628:Gene Dosage; D020131:Genes, Duplicate; D005838:Genotype; D015417:Hereditary Sensory and Motor Neuropathy / Q000235:genetics*; D006801:Humans; D009185:Myelin Proteins / Q000235:genetics; D010243:Paralysis / Q000235:genetics; D016133:Polymerase Chain Reaction
D009185:Myelin Proteins; C073687:PMP22 protein, human; D004247:DNA