Allele Drop-Out in the MECP2 Gene Due to G-Quadruplex and i-Motif Sequences When Using Polymerase Chain Reaction-Based Diagnosis for Rett Syndrome

Abstract

Although few examples are formally documented, all polymerase chain reaction-based testing is theoretically vulnerable to allele drop-out (ADO), the failure to amplify one of the two alleles present in a cell. In a clinical setting, this can lead to false positive or negative diagnosis. We investigated the mechanisms leading to ADO in the MECP2 gene in two unrelated female patients undergoing testing for Rett syndrome. Both the patients had two benign DNA variations, c.819G > T and c.1161C > T, that appeared homozygous due to ADO. Bioinformatics analyses indicate that this region of the MECP2 gene is rich in complex tertiary structures called G-quadruplex and i-motifs, the disruption of which by the c.819G > T and c.1161C > T variants leads to preferential amplification of the variant allele. Other examples of ADO likely occur, and consideration of disrupting G-quadruplex and i-motif structures should be given when this phenomenon is unexpected. We identify factors in both the polymerase chain reaction amplification and the sequencing steps that help overcome ADO.

The Molecular Bases of Phenylketonuria (PKU) in New South Wales, Australia: Mutation Profile and Correlation with Tetrahydrobiopterin (BH4) Responsiveness

“…Pathogenic mutations were not found in only two of the alleles tested. Polymorphisms in the location of the PCR primers may lead to the drop-out of an allele during amplification, as well as complex tertiary structures in the DNA and poor template quality or quantity (Tvedebrink et al 2009;Saunders et al 2010). Alternate primer sets or whole exome/genome sequencing may be of use in the identification of the second alleles in these patients, as mutations further into the intron or in the promoter or untranslated regions were not investigated in this study.…”

Structural Analysis of G-Quadruplex Formation at the Human MEST Promoter

“…Although such structures are now recognized to have many biological functions [2–11], they can also present problems for the in vitro analysis of DNA by polymerase chain reaction (PCR) and related techniques [12–14]. Previously, we reported persistent allelic drop-out (ADO) during polymerase chain reaction (PCR) analysis of the human MEST promoter region (Fig 1).…”

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

“…Despite extensive optimization and near ubiquitous usage, PCR is still prone to failure under certain circumstances. For diploid organisms, the failure of one allele to amplify can result in allelic dropout (ADO), causing apparent homozygosity (Askree et al 2011; Boán et al 2004; Lam and Mak 2013; Landsverk et al 2012; Piyamongkol et al 2003; Saunders et al 2010; Wenzel et al 2009). ADO is an insidious problem that is difficult to recognize because the PCR appears successful, but half of the genetic information is missing.…”

“…ADO can have significant implications in both research and clinical applications, where there is a requirement for high sensitivity and accurate PCR genotyping. Incorrect genotyping can have substantial negative consequences and may result in the misdiagnosis of genetic diseases, loss of the ability to differentiate between individuals, and false assumptions about parentage or genetic diversity (Boán et al 2004; Landsverk et al 2012; Saunders et al 2010; Tomaz et al 2010; Wenzel et al 2009). …”

“…G4 formation is stabilized by the integration of a cation, like potassium (Ambrus et al 2005; Biffi et al 2012; Maizels 2015; Rhodes and Lipps 2015; Simonsson 2001), making PCR buffer an optimal environment for G4 formation. G4 structures may then act as a steric block to Taq polymerase (Boán et al 2004; Chambers et al 2015; Saunders et al 2010; Weitzmann et al 1996), an effect that is exacerbated when the G4 region is methylated (Stevens et al 2014). …”