Detection of three closely located single nucleotide polymorphisms in the EAAT2 promoter: comparison of single-strand conformational polymorphism (SSCP), pyrosequencing and Sanger sequencing

Abstract

BackgroundSingle-strand conformational polymorphism (SSCP) is still a frequently used genotyping method across different fields for the detection of single nucleotide polymorphisms (SNPs) due to its simplicity, requirement for basic equipment accessible in most laboratories and low cost. This technique was previously used to detect rs4354668:A > C (g.-181A > C) SNP in the promoter of astroglial glutamate transporter (EAAT2) and the same approach was initially used here to investigate this promoter region in a cohort of newborns.ResultsUnexpectedly, four distinct DNA migration patterns were identified by SSCP. Sanger sequencing revealed two additional SNPs: g.-200C > A and g.-168C > T giving a rise to a total of ten EAAT2 promoter variants. SSCP failed to distinguish these variants reliably and thus pyrosequencing assays were developed. g.-168C > T was found in heterozygous form in one infant only with minor allele frequency (MAF) of 0.0023. In contrast, g.-200C > A and -181A > C were more common (with MAF of 0.46 and 0.49, respectively) and showed string evidence of linkage disequilibrium (LD). In a systematic comparison, 16% of samples were miss-classified by SSCP with 25-31% errors in the identification of the wild-type and homozygote mutant genotypes compared to pyrosequencing or Sanger sequencing. In contrast, SSCP and pyrosequencing of an unrelated single SNP (rs1835740:C > T), showed 94% concordance.ConclusionOur data suggest that SSCP cannot always detect reliably several closely located SNPs. Furthermore, caution is needed in the interpretation of the association studies linking only one of the co-inherited SNPs in the EAAT2 promoter to human diseases.

Variants of the EAAT2 Glutamate Transporter Gene Promoter Are Associated with Cerebral Palsy in Preterm Infants

“…Amplification was performed as follows: 95 °C for 5 min, 50 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and final extension 72 °C for 10 min. Two additional SNPs, rs116392274 in EAAT2 and rs1835740 [ 21 ], which are involved in glutamate homeostasis, were also analysed in the cohort and data are shown as Supplementary materials .…”

“…These studies raised the intriguing possibility that similar genetic differences may enhance predisposition to neurodevelopmental impairment after preterm birth. The aim of this study was to establish the role of two closely linked functional SNPs in the EAAT2 gene promoter [ 19 , 21 ] in susceptibility to brain injury and neurodisability in very preterm infants.…”

Quantitative analysis of single‐nucleotide polymorphisms by pyrosequencing with di‐base addition

“…The generated visible light is recorded as a series of peaks called a pyrogram, which represents the order of complementary dNTPs and implies the underlying DNA sequence . Pyrosequencing has well suited the analysis of SNPs , mutations , and DNA methylations in the target samples. It offers an easy, quantitative, and accurate method to detect the relative abundance of alleles in a mixture.…”

New markers of atherosclerosis: a threshold level of heteroplasmy in mtDNA mutations

“…The study was carried out using amplifier "PTC DNA Engine 200". After that pyrosequencing of PCR fragments was conducted using the apparatus (PSQ96MA) [30][31][32][33][34] . The pyrosequencing reaction included 4 stages [30][31][32][33][34] .…”