Effectiveness of Real-Time Quantitative PCR Compare to Repeat PCR for CMT1A and

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Yonsei Med J. 2005 Jun 30;46(3):347-52.

(NOTE: This is about faster, more accurate DNA testing for CMT1A and HNPP ~

Gretchen)

Effectiveness of Real-Time Quantitative PCR Compare to Repeat PCR for

the Diagnosis of Charcot-Marie-Tooth Type 1A and Hereditary

Neuropathy with Liability to Pressure Palsies.

Choi JR, Lee WH, Sunwoo IN, Lee EK, Lee CH, Lim JB.

Departments of Laboratory Medicine, Yonsei University College of

Medicine, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-752, Korea.

The majority of cases of Charcot-Marie-Tooth type 1A (CMT1A) and of

hereditary neuropathy with a liability to pressure palsies (HNPP) are

the result of heterozygosity for the duplication or deletion of

peripheral myelin protein 22 gene (PMP22) on 17p11.2. Southern blots,

pulsed-field gel electrophoresis (PFGE), fluorescence in situ

hybridization (FISH) and polymorphic marker analysis are currently

used diagnostic methods. But they are time-consuming, labor-intensive

and have some significant limitations.

We describe a rapid real- time quantitative PCR method for

determining gene copy number for the identification of DNA

duplication or deletion occurring in CMT1A or HNPP and compare the

results obtained with REP-PCR.

Six patients with CMT1A and 14 patients with HNPP [confirmed by

Repeat (REP)-PCR], and 16 patients with suspicious CMT1A and 13

patients with suspicious HNPP [negative REP-PCR], and 15 normal

controls were studied. We performed REP-PCR, which amplified a 3.6 Kb

region (including a 1.7Kb recombination hotspot), using specific

CMT1A-REP and real-time quantitative PCR on the LightCycler system.

Using a comparative threshold cycle (Ct) method and beta -globin as a

reference gene, the gene copy number of the PMP22 gene was

quantified. The PMP22 duplication ratio ranged from 1.35 to 1.74, and

the PMP22 deletion ratio from 0.41 to 0.53. The PMP22 ratio in normal

controls ranged from 0.81 to 1.12.

All 6 patients with CMT1A and 14 patients with HNPP confirmed by REP-

PCR were positive by real-time quantitative PCR.

Among the 16 suspicious CMT1A and 13 suspicious HNPP with negative

REP-PCR, 2 and 4 samples, respectively, were positive by real-time

quantitative PCR.

Real-time quantitative PCR is a more sensitive and more accurate

method than REP-PCR for the detection of PMP22 duplications or

deletions, and it is also faster and easier than currently available

methods. Therefore, we believe that the real-time quantitative method

is useful for diagnosing CMT1A and HNPP.