ORIGINAL ARTICLES Quantitative Fluorescence-Polymerase Chain Reaction Assay for the Detection of the Duplication of the Charcot Marie Tooth Disease Type 1A Critical Region Simona De Toffol, 1 Emilia Bellone, 2 Francesca Dulcetti, 1 Anna Maria Ruggeri, 1 Pietro Paolo Maggio, 3 Maria Rosaria Pulimeno, 4 Paola Mandich, 2 Federico Maggi, 1 Giuseppe Simoni, 1 and Francesca Romana Grati 1 Charcot Marie tooth (CMT) syndrome is the most common hereditary peripheral neuropathy, with an incidence of about 1 in 2500. The subtype 1A (CMT1A) is caused by a tandem duplication of a 1.5-Mb region encom- passing the PMP22 gene. Conventional short tandem repeat (STR) analysis can reveal this unbalance if a triallelic pattern, defining with certainty the presence of duplication, is present. In case of duplication with a biallelic pattern, it can only indicate a semiquantitative dosage of the fluorescence intensity ratio of the two fragments. In this study we developed a quantitative fluorescence-PCR using seven highly informative STRs within the CMT1A critical region that successfully disclosed or excluded the presence of the pathogenic unbalance in a cohort of 60 samples including 40 DNAs from samples with the CMT1A duplication previously characterized with two different molecular approaches, and 20 diagnostic samples from 10 members of a five-generation pedigree segregating CMT1A (8 unrelated cases and 2 prenatal samples). The application of the quantitative fluorescence-PCR using STRs located in the critical region could be a reliable method to evaluate the presence of the PMP22 duplication for the diagnosis and classification of hereditary neuropathies in asymptomatic subjects with a family history of inherited neuropathy, in prenatal samples in cases with one affected parent, and in unrelated patients with a sporadic demyelinating neuropathy with clinical features resembling CMT (i.e., pes cavus with hammer toes) or with conduction velocities in the range of CMT1A. Introduction AU2 c C harcot Marie tooth (CMT) is the most common he- reditary sensory and motor peripheral neuropathy (1in 2500). It is a clinically and genetically heterogeneous disease. CMT has been linked to 36 loci and mutations have been identified in 28 different genes; the phenotype of CMT is ex- tremely variable, ranging from a severe polyneuropathy with respiratory failure through the classic picture with pes cavus and ‘‘stork legs’’ to minimal neurological findings (Skre, 1974; England et al., 2009). It is mostly inherited as an autosomal dominant trait, but X-linked, autosomal recessive and spo- radic cases are also found. Two major forms can be distin- guished: type 1 (CMT1) or demyelinating form shows defects in the formation and maintenance of myelin; type 2 (CMT2) or axonal form is characterized by axonal degeneration (Harding and Thomas, 1980; England et al., 2009). The demyelinating form of CMT1 is the most prevalent and the type 1A (CMT1A, MIM no. 118220) account for about 70% of all cases of CMT1 (Nelis et al., 1996; Leonardis et al., 1998; Nicholson, 1999; Mersiyanova et al., 2000; Mostacciuolo et al., 2001; Choi et al., 2004). CMT1A is also the most common va- riety of sporadic CMT1, accounting for 76–90% of cases (England et al., 2009). The vast majority of CMT1A cases (>98%) have a hetero- zygous tandem duplication of a 1.5-Mb region encompassing peripheral myelin protein-22 (PMP22, 17p11.2–p12), which plays an important role in the function of the human pe- ripheral nervous system (Lupski et al., 1991). The duplication leading to CMT1A occurs mainly during spermatogenesis and is caused by unequal crossing over between two 24-kb homologous repetitive elements (proximal and distal CMT1A-REPs b AU3 ) flanking PMP22 (Pentao et al., 1992; Yama- moto et al., 1998). Rare cases (2.5%) carry a point mutation in 1 b AU1 Unit of Research and Development, Cytogenetics, and Molecular Biology, TOMA Advanced Biomedical Assays S.p.A., Busto Arsizio, Varese, Italy. 2 Section of Medical Genetics, Department of Neuroscience, Ophthalmology, and Genetics, University of Genoa, Genoa, Italy. 3 Cytogenetics and Molecular Biology U.O. and 4 Neurology U.O., ‘‘Sacro Cuore di Gesu ` ’’ Hospital, Gallipoli, Lecce, Italy. GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 14, Number 2, 2010 ª Mary Ann Liebert, Inc. Pp. 1–7 DOI: 10.1089=gtmb.2009.0118 1 GTMB-2009-0118-Toffol_1P Type: research-article GTMB-2009-0118-Toffol_1P.3D 01/18/10 3:21pm Page 1