A simple method for examination of polymorphisms of catalase exon 9: rs769217 in Hungarian microcytic anemia and beta-thalassemia patients

• Published in: Archives of biochemistry and biophysics 2012-09, Vol.525 (2), p.201-206
• Main Authors: Nagy, Teréz, Csordás, Melinda, Kósa, Zsuzsanna, Góth, László
• Format: Article
• Language: English
• Subjects: Acatalasemia; Adolescent; Adult; anemia; Anemia - ethnology; Anemia - genetics; Beta-thalassemia; beta-Thalassemia - ethnology; beta-Thalassemia - genetics; blood; catalase; Catalase - genetics; Catalase - metabolism; Catalase gene mutations screening; denaturation; digestion; DNA; ethidium; Exons; Female; gel electrophoresis; Genotype; Humans; Hungary; hydrogen peroxide; Male; Microcytic anemia; Middle Aged; Models, Genetic; Mutation; patients; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Polymorphism, Single-Stranded Conformational; restriction fragment length polymorphism; screening; sequence analysis; Sequence Analysis, DNA - methods; silver; single-stranded conformational polymorphism; SSCP; toxicity
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2012.01.004

[Display omitted] ► Electrophoresis of PCR amplicons of catalase exon 9 showed unusual bands in 400–800bp region. ► They might be further existing single strands due to internal base pairing. ► Their unique electrophoretic pattern may be used for examination of polymorphisms in catalase exon 9. Catalase decreases the high, toxic concentrations of hydrogen peroxide but it lets the physiological, low concentrations in the cells mainly for signaling purposes. Its decreased activity may contribute to development of several pathological conditions. Catalase mutations occur frequently in exon 9, these were examined with different, complicated and costly methods. The aim of the current study was to evaluate a method for screening of polymorphisms in catalase exon 9. We used the slab gel electrophoresis of PCR amplicons without denaturation and silver staining for visualization of the DNA bands. We detected extra DNA bands in the 400–800bp region of the catalase exon 9. Their single stranded nature was proved with nucleotide sequence analyses, comparison with the standard SSCP, staining with Sybr Green II and Sybr Green I, ethidium bromide, no digestion with RFLP (BstX I), and digestion with plant nuclease. We used this method for examination of polymorphisms of catalase exon 9 in microcytic anemia and beta-thalassemia patients. The lowest blood catalase activities were detected in microcytic anemia and beta-thalassemia patients with the TT genotypes of the C111T polymorphism. This method was sensitive for detection of G113A acatalasemia mutation, but poorly detected C37T and G5A acatalasemia mutations.