Abstract
Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5-Mb duplication and a deletion at chromosome 17p11.2–12 encompassing the peripheral myelin protein 22 gene (PMP22), respectively. We developed a rapid and reliable detection system for duplications/deletions of the PMP22 gene based on measurement of gene copy number. The method involves amplification of a test locus with unknown copy number and a reference locus of known copy number by multiplex PCR (MP), followed by denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, 17 patients with HNPP, and 61 unaffected individuals were analyzed. Using the same competitive MP protocol, the measured PMP22 gene dosage revealed concordant results between DHPLC and capillary electrophoresis analysis. The results of the MP/DHPLC or the MP/capillary electrophoresis assay were all confirmed by PCR–restriction fragment length polymorphism analysis. We concluded that the MP/DHPLC assay is an efficient, accurate, and reliable technique for gene dosage determination of the PMP22 gene for CMT1A duplication and HNPP deletion. This technique further extends the application of DHPLC as an alternative method for the measurement of gene amplifications and heterozygous deletions in different genetic diseases.
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References
Akrami SM, Rowland JS, Taylor GR, Armour JA (2003) Diagnosis of gene dosage alterations at the PMP22 gene using MAPH. J Med Genet 40(11):e123
Badano JL, Inoue K, Katsanis N, Lupski JR (2001) New polymorphic short tandem repeats for PCR-based Charcot-Marie-Tooth disease type 1A duplication diagnosis. Clin Chem 47:838–843
Beckmann A, Schroder JM (2000) Screening for Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsy in archival nerve biopsy samples by direct-double-differential PCR. Acta Neuropathol 100:459–463
Boerkoel CF, Takashima H, Garcia CA, Olney RK, Johnson J, Berry K, Russo P, Kennedy S, Teebi AS, Scavina M et al (2002) Charcot-Marie-Tooth disease and related neuropathies: mutation distribution and genotype–phenotype correlation. Ann Neurol 51:190–201
Celi FS, Zenilman ME, Shuldiner AR (1993) A rapid and versatile method to synthesize internal standards for competitive PCR. Nucleic Acids Res 21:1047
Chance PF, Alderson MK, Leppig KA, Lensch MW, Matsunami N, Smith B, Swanson PD, Odelberg SJ, Disteche CM, Bird TD (1993) DNA deletion associated with hereditary neuropathy with liability to pressure palsies. Cell 72:143–151
Dehainault C, Lauge A, Caux-Moncoutier V, Pages-Berhouet S, Doz F, Desjardins L, Couturier J, Gauthier-Villars M, Stoppa-Lyonnet D, Houdayer C (2004) Multiplex PCR/liquid chromatography assay for detection of gene rearrangements: application to RB1 gene. Nucleic Acids Res 32:e139
Frisso G, Carsana A, Tinto N, Calcagno G, Salvatore F, Sacchetti L (2004) Direct detection of exon deletions/duplications in female carriers of and male patients with Duchenne/Becker muscular dystrophy. Clin Chem 50:1435–1438
Ikegami T, Ikeda H, Chance PF, Kiyosawa H, Yamamoto M, Sobue G, Ohnishi A, Tachi N, Hayasaka K (1997) Facilitated diagnosis of CMT1A duplication in chromosome 17p11.2–12: analysis with a CMT1A-REP repeat probe and photostimulated luminescence imaging. Hum Mutat 9:563–566
Kim SW, Lee KS, Jin HS, Lee TM, Koo SK, Lee YJ, Jung SC (2003) Rapid detection of duplication/deletion of the PMP22 gene in patients with Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsy by real-time quantitative PCR using SYBR Green I dye. J Korean Med Sci 18:727–732
Latour P, Boutrand L, Levy N, Bernard R, Boyer A, Claustrat F, Chazot G, Boucherat M, Vandenberghe A (2001) Polymorphic short tandem repeats for diagnosis of the Charcot-Marie-Tooth 1A duplication. Clin Chem 47:829–837
Laurendeau I, Bahuau M, Vodovar N, Larramendy C, Olivi M, Bieche I, Vidaud M, Vidaud D (1999) TaqMan PCR-based gene dosage assay for predictive testing in individuals from a cancer family with INK4 locus haploinsufficiency. Clin Chem 45:982–986
Lupski JR, de Oca-Luna RM, Slaugenhaupt S, Pentao L, Guzzetta V, Trask BJ, Saucedo-Cardenas O, Barker DF, Killian JM, Garcia CA et al (1991) DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 66:219–232
Lynch M (2002) Genomics. Gene duplication and evolution. Science 297:945–947
MacMillan JC, Upadhyaya M, Harper PS (1992) Charcot-Marie-Tooth disease type 1a (CMT1a): evidence for trisomy of the region p11.2 of chromosome 17 in south Wales families. J Med Genet 29:12–13
Navon R, Timmerman V, Lofgren A, Liang P, Nelis E, Zeitune M, Van Broeckhoven C (1995) Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A (CMT1A) using molecular genetic techniques. Prenat Diagn 15:633–640
Noonan KE, Beck C, Holzmayer TA, Chin JE, Wunder JS, Andrulis IL, Gazdar AF, Willman CL, Griffith B, Von Hoff DD et al (1990) Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. Proc Natl Acad Sci USA 87:7160–7164
Pan CL, Tseng TJ, Lin YH, Chiang MC, Lin WM, Hsieh ST (2003) Cutaneous innervation in Guillain-Barre syndrome: pathology and clinical correlations. Brain 126:386–397
Patel PI, Roa BB, Welcher AA, Schoener-Scott R, Trask BJ, Pentao L, Snipes GJ, Garcia CA, Francke U, Shooter EM et al (1992) The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A. Nat Genet 1:159–165
Pentao L, Wise CA, Chinault AC, Patel PI, Lupski JR (1992) Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit. Nat Genet 2:292–300
Poropat RA, Nicholson GA (1998) Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR. Clin Chem 44:724–730
Raeymaekers P, Timmerman V, Nelis E, De Jonghe P, Hoogendijk JE, Baas F, Barker DF, Martin JJ, De Visser M, Bolhuis PA, et al (1991) Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1a (CMT 1a). The HMSN Collaborative Research Group. Neuromuscul Disord 1:93–97
Roa BB, Greenberg F, Gunaratne P, Sauer CM, Lubinsky MS, Kozma C, Meck JM, Magenis RE, Shaffer LG, Lupski JR (1996) Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1 neuropathy. Hum Genet 97:642–649
Rowland JS, Barton DE, Taylor GR (2001) A comparison of methods for gene dosage analysis in HMSN type 1. J Med Genet 38:90–95
Skre H (1974) Genetic and clinical aspects of Charcot-Marie-Tooth’s disease. Clin Genet 6:98–118
Stronach EA, Clark C, Bell C, Lofgren A, McKay NG, Timmerman V, Van Broeckhoven C, Haites NE (1999) Novel PCR-based diagnostic tools for Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsies. J Peripher Nerv Syst 4:117–122
Su YN, Hung CC, Li H, Lee CN, Cheng WF, Tsao PN, Chang MC, Yu CL, Hsieh WS, Lin WL, et al (2005) Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a highly efficient and reliable carrier-screening test. Hum Mutat 25:460–467
Thiel CT, Kraus C, Rauch A, Ekici AB, Rautenstrauss B, Reis A (2003) A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2–12 duplications and deletions leading to HMSN/HNPP. Eur J Hum Genet 11:170–178
Timmerman V, Nelis E, Van Hul W, Nieuwenhuijsen BW, Chen KL, Wang S, Ben Othman K, Cullen B, Leach RJ, Hanemann CO et al (1992) The peripheral myelin protein gene PMP-22 is contained within the Charcot-Marie-Tooth disease type 1A duplication. Nat Genet 1:171–175
Timmerman V, Lofgren A, Le Guern E, Liang P, De Jonghe P, Martin JJ, Verhalle D, Robberecht W, Gouider R, Brice A et al (1996) Molecular genetic analysis of the 17p11.2 region in patients with hereditary neuropathy with liability to pressure palsies (HNPP). Hum Genet 97:26–34
Wilke K, Duman B, Horst J (2000) Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR. Hum Mutat 16:431–436
Yau SC, Bobrow M, Mathew CG, Abbs SJ (1996) Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis. J Med Genet 33:550–558
Young P, Stogbauer F, Wiebusch H, Lofgren A, Timmerman V, Van Broeckhoven C, Ringelstein EB, Assmann G, Funke H (1998) PCR-based strategy for the diagnosis of hereditary neuropathy with liability to pressure palsies and Charcot-Marie-Tooth disease type 1A. Neurology 50:760–763
Zhu D, Kennerson M, Merory J, Chrast R, Verheijen M, Lemke G, Nicholson G (2003) Refined localization of dominant intermediate Charcot-Marie-Tooth neuropathy and exclusion of seven known candidate genes in the region. Neurogenetics 4:179–183
Acknowledgment
This work was supported by Grants from the National Science Council of Taiwan (NSC 93-2314-B-002 174).
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Chia-Yun Lin and Yi-Ning Su share the first authorship.
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Lin, CY., Su, YN., Lee, CN. et al. A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay. J Hum Genet 51, 227–235 (2006). https://doi.org/10.1007/s10038-005-0350-9
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DOI: https://doi.org/10.1007/s10038-005-0350-9
