Detection of a 1,5 Mb duplication or deletion in the chromosomal region 17p11.2-p12 by
real-time PCR
Authors: Reško Péter1, Radvánszky János1, Pálffy Roland2
Tutors: RNDr. Helena Poláková, RNDr1. Kádasi Lajos, PhD.1
1
Institute: Commenius University, Faculty of Natural Sciences, Department of Molecular
Biology, Bratislava
2
Commenius University, Faculty of Medicine, Institute of Pathophysiology,
Bratislava
The aim of this study was the optimization of reaction conditions for the precise
quantification of peripheral myelin protein 22 gene (PMP22) by Real-time PCR.
Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with
liability to pressure palsies (HNPP) are two distinct types of inherited peripheral neuropathies
with dominant inheritance. 70% of CMT1A and 80% of HNPP cases are results of DNA
duplication and deletion respectively of a 1,5 megabase (Mb) region on chromosome 17p11.2.
This chromosomal region contains the PMP22 gene. The molecular cause of CMT1A and
HNPP in patients with duplication and deletion of PMP22 is the dosage sensitivity of this
gene, thus genetic tests for screening of these patients are based on the quantification of gene
copy number of PMP22.
Real-time PCR have been used to quantify the PMP22 gene copy number in this study,
because it’s one of the best approaches for nucleic acid quantification today. In addition, two
different types of PCR product visualization (probe based and intercalator based) and two
different real-time instruments (ABI PRISM 7900HT Sequence Detection System and
MiniOpticon Real-Time PCR System) have been compared. The results obtained by real-time
PCR were approved by gel electrophoresis of the PCR products.
Reaction conditions of real-time PCR for the quantification of PMP22 using TaqMan
probes and ABI PRISM 7900HT Sequence Detection System were successfully optimized.
This method of gene copy number quantification seems to be more sensitive, more reliable
and faster than all methods used for PMP22 quantification until now and reduces the
probability of cross contamination. In contrast, the optimization of reaction conditions using
MiniOpticon Real-Time PCR System was not successful, thus ABI PRISM 7900HT Sequence
Detection System seems to be more suitable for real-time quantification of PMP22 in
combination with both probe based and intercalator based dyes.
Keywords: CMT1A, HNPP, PMP22, gene copy number quantification, real-time PCR.