title: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, The Children's Mercy Hospitals and Clinics, Kansas City, Missouri 64108, USA. csaunders@cmh.edu
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Saunders
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Friez
authors.forename: Melanie
authors.initials: M
authors.lastname: Patterson
authors.forename: Masha
authors.initials: M
authors.lastname: Nzabi
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhao
authors.forename: Chengpeng
authors.initials: C
authors.lastname: Bi
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002648:Child; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005260:Female; D054856:G-Quadruplexes; D005820:Genetic Testing; D006720:Homozygote; D006801:Humans; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D015518:Rett Syndrome / Q000175:diagnosis* / Q000235:genetics*
chemical_list: D017931:DNA Primers; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D004247:DNA