Type 1A CMT + HNPP research - from Germany

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Abstract from European Journal of Human Genetics 2003 Feb;11(2):170-8

A new quantitative PCR multiplex assay for rapid analysis of chromosome

17p11.2-12 duplications and deletions leading to HMSN/HNPP.

Thiel CT, Kraus C, Rauch A, Ekici AB, Rautenstrauss B, Reis A.

Institute of Human Genetics, University of Erlangen-Nuremberg,

Schwabachanlage 10, 91054 Erlangen, Germany.

A 1.4-Mb tandem duplication, including the gene for peripheral myelin

protein 22 (PMP22) in chromosome 17p11.2-12 is responsible for 70% of

the cases of the demyelinating type 1 of Charcot-Marie-Tooth disease or

hereditary motor and sensory neuropathy I (CMT1A/HMSN I). A reciprocal

deletion of this CMT1A region causes the hereditary neuropathy with

liability to pressure palsies (HNPP). The CMT1A duplication increases

the PMP22 gene dosage from two to three, the HNPP deletion reduces the

gene dosage from two to one. Currently, routine diagnosis of HMSN/HNPP

patients is mainly performed with polymorphic markers in-between the

repetitive elements flanking the

CMT1A region. These show quantitative and/or qualitative changes in case

of a CMT1A duplication and a homozygous allele pattern in case of HNPP

deletion. In HNPP patients the deletion is usually confirmed by

fluorescence in situ hybridisation (FISH). We now developed a reliable,

single tube real-time quantitative PCR assay for rapid determination of

PMP22 gene dosage directly. This method involves a multiplex reaction

using FAM labelled Taqman-probe with TAMRA quencher derived from PMP22

exon 3 and a VIC labelled probe with non-fluorescent quencher from exon

12 of the albumin gene as internal reference. Copy number of the PMP22

gene was determined by the comparative threshold cycle method

(DeltaDeltaCt). Each sample was run in quadruplicate and

analysed at two different threshold levels. The level giving the

smallest standard deviation was scored. We evaluated this method through

the retrospective analysis of 252 HMSN patients with known genotype and

could confirm the previous findings in 99% of cases. Two patients were

wrongly diagnosed with microsatellite analysis while quantitative

real-time PCR identified the correct genotype, as confirmed by FISH.

Thus, this method shows superior sensitivity to microsatellite analysis

and has the additional advantage of being a fast and uniform assay for

quantitative analysis of both CMT1A and HNPP.