title: G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.
abstract: We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Stevens
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Selma
authors.initials: S
authors.lastname: Stuffrein-Roberts
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Simone L
authors.initials: SL
authors.lastname: Cree
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Andrew
authors.initials: A
authors.lastname: Gibb
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Allison L
authors.initials: AL
authors.lastname: Miller
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Kit
authors.initials: K
authors.lastname: Doudney
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Alan
authors.initials: A
authors.lastname: Aitchison
authors.affiliation: Department of Pathology, University of Otago, Dunedin School of Medicine, Dunedin, New Zealand.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Eccles
authors.affiliation: Department of Psychological Medicine, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Joyce
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Vyacheslav V
authors.initials: VV
authors.lastname: Filichev
authors.affiliation: Department of Pathology, University of Otago, Christchurch 8140, New Zealand.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Kennedy
mesh_terms: D000483:Alleles; D002942:Circular Dichroism; D018899:CpG Islands; D019175:DNA Methylation; D003865:Major Depressive Disorder / Q000235:genetics*; D054856:G-Quadruplexes; D018392:Genomic Imprinting; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods; D008958:Models, Molecular; D016133:Polymerase Chain Reaction / Q000379:methods*; D011188:Potassium / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D011506:Proteins; C103637:mesoderm specific transcript protein; D011188:Potassium