Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR

We report the development of a method fur diagnosis of heterozygous deletio ns or duplications based on measurement of gene copy number. The method inv olves amplifications of a test locus with unknown copy number and a referen ce locus with known copy number using real-time PCR. Progress of the PCR re actions is monitored using fluorigenic probes and a real-time fluorescence detection system. For each reaction, the number of cycles is measured at wh ich a defined thresh old fluorescence emission is reached. Using standard c urves, the copy number of the test DNA relative to a common standard DNA is determined for each locus. From the ratio of the relative copy numbers, th e genomic copy number of the test locus is determined. In order to demonstr ate the accuracy and reliability of the method for genetic testing, we anal yzed 43 patients with hereditary neuropathy with liability to pressure pals ies (HNPP), containing a heterozygous deletion of a 1.5 Mb region on chromo some 17p11.2-p12, eight patients with Charcot-Marie-Tooth disease, containi ng a heterozygous duplication of the same genomic region, and 50 normal con trol individuals. As a test locus we analyzed the PMP22 gene located within the 1.5 Mb region. The genomic copy number of the test locus was precisely measured, and the presence or absence of the genomic deletion or duplicati on was unambiguously diagnosed in all individuals. Hum Mutat 16:431-436, 20 00. (C) 2000 Wiley-Liss, Inc.