Dataset Information

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

ABSTRACT: Single-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.Unexpectedly, 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.Our 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.

SUBMITTER: Rajatileka S

PROVIDER: S-EPMC4112986 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Rajatileka Shavanthi S Luyt Karen K Williams Maggie M Harding David D Odd David D Molnár Elek E Váradi Anikó A

BMC genetics 20140705

<h4>Background</h4>Single-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 th ...[more]

PMID: 24996834

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Project description:BACKGROUND:We have previously developed a long RT-PCR method for selective amplification of full-length PKD1 transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the PKD1 gene. These have provided us with an opportunity to study PKD1 mutations especially in its reiterated region which is difficult to examine. In this report, we have further developed the method of multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) for analysis of PKD1 mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and de novo PKD1 mutations are identified and reported. METHODS:Full-length PKD1 cDNA isolated from the patients with ADPKD was fractionated into nine overlapping segments by nested-PCR. Each segment was digested with sets of combined restriction endonucleases before the SSCP analysis. The fragments with aberrant migration were mapped, isolated, and sequenced. The presence of mutation was confirmed by the long-range genomic DNA amplification in the PKD1 region, sequencing, direct mutation detection, and segregation analysis in the affected family. RESULTS:Five PKD1 mutations identified are two frameshift mutations caused by two di-nucleotide (c. 5225_5226delAG and c.9451_9452delAT) deletions, a nonsense (Q1828X, c.5693C>T) mutation, a splicing defect attributable to 31 nucleotide deletion (g.33184_33214del31), and an in-frame deletion (L3287del, c.10070_10072delCTC). All mutations occurred within the reiterated region of the gene involving exons 15, 26, 15, 19 and 29, respectively. Three mutations (one frameshift, splicing defect, and in-frame deletion) are novel and two (one frameshift and nonsense) known. In addition, two mutations (nonsense and splicing defect) are possibly de novo. CONCLUSION:The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening PKD1 mutations in the full-length cDNA. Five mutations identified were all in the reiterated region of this gene, three of which were novel. The presence of de novo PKD1 mutations indicates that this gene is prone to mutations.

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Dataset Information

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

Publications

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

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