title: Multiplex ligation-dependent probe amplification for rapid detection of proteolipid protein 1 gene duplications and deletions in affected males and carrier females with Pelizaeus-Merzbacher disease.
abstract: BACKGROUND
  Pelizaeus-Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for approximately 50%-75% of cases and point variations for approximately 15%-20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions.
  
  
  METHODS
  We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex.
  
  
  RESULTS
  All males with PLP1 gene duplications (n = 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n = 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. For 1 sample with a partial deletion, MLPA showed exon 3 deleted but PCR showed exons 3 and 4 deleted. Sequence analysis of 2 samples with reduced dosage for exons 3 and 5 revealed point variations overlapping the annealing site for the corresponding MLPA probe. The precision of MLPA analysis was excellent and comparable to or better than quantitative PCR, with CVs of 4.3%-9.8%.
  
  
  CONCLUSIONS
  MLPA is a rapid and reliable method to determine PLP1 gene copies. Samples with partial PLP1 gene dosage alterations require confirmation with a non-MLPA method.
authors.affiliation: Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. warshai@ccf.org
authors.forename: Ilka
authors.initials: I
authors.lastname: Warshawsky
authors.forename: Olga B
authors.initials: OB
authors.lastname: Chernova
authors.forename: Christian A
authors.initials: CA
authors.lastname: Hübner
authors.forename: Reinhard
authors.initials: R
authors.lastname: Stindl
authors.forename: Marco
authors.initials: M
authors.lastname: Henneke
authors.forename: Andreas
authors.initials: A
authors.lastname: Gal
authors.forename: Marvin R
authors.initials: MR
authors.lastname: Natowicz
mesh_terms: D002353:Carrier State; D005260:Female; D017353:Gene Deletion; D020440:Gene Duplication; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D018991:Myelin Proteolipid Protein / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D020371:Pelizaeus-Merzbacher Disease / Q000235:genetics*; D012016:Reference Values
chemical_list: D008565:Membrane Proteins; D018991:Myelin Proteolipid Protein; D015345:Oligonucleotide Probes; C472930:PLP1 protein, human