G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter

• Published in: PloS one Vol. 9; no. 12; p. e113955
• Main Authors: Stevens, Aaron J, Stuffrein-Roberts, Selma, Cree, Simone L, Gibb, Andrew, Miller, Allison L, Doudney, Kit, Aitchison, Alan, Eccles, Michael R, Joyce, Peter R, Filichev, Vyacheslav V, Kennedy, Martin A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01-12-2014; Public Library of Science (PLoS)
• Subjects: Alleles; Amplification; Analysis; Biology and life sciences; Book publishing; Circular Dichroism; CpG Islands; Deoxyribonucleic acid; Depressive Disorder, Major - genetics; Diagnostic systems; Dichroism; DNA; DNA Methylation; Electrophoresis; Epigenetics; G-Quadruplexes; Gel electrophoresis; Gene expression; Genes; Genetic testing; Genomes; Genomic Imprinting; Genomics; Genotyping; Haplotypes; Human populations; Humans; In Vitro Techniques - methods; Linkage disequilibrium; Mental depression; MEST gene; Methylation; Models, Molecular; Pathology; Polymerase chain reaction; Polymerase Chain Reaction - methods; Potassium; Potassium - chemistry; Primers; Promoter Regions, Genetic; Proteins - chemistry; Proteins - genetics; Research and Analysis Methods; Sequence Analysis, DNA; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Spectroscopy; New Zealand
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0113955

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.