Detection of single nucleotide polymorphisms in coagulation factor XI deficient patients by multitemperature single-strand conformation polymorphism analysis

Abstract

Factor XI (FXI) deficiency is a rare inherited disorder which can cause bleeding complications especially in case of hemostatic challenge and/or in tissues with high fibrinolytic activity. A number of causative mutations have been described in FXI deficient individuals which have been detected by various screening methods. In this study, we present the application of the multitemperature single-strand conformation polymorphism analysis (MSSCP) on the FXI gene, a recently developed methodology for the detection of single nucleotide exchanges. We analyzed a total of 217 polymerase chain reaction (PCR) fragments from the promoter region as well as from exons 1-7 and 11-15 and compared the results to automatic fluorescent sequencing. A total of 29 PCR fragments showed single nucleotide exchanges in conventional fluorescent sequencing, representing 10 different mutations (nine missense mutations, one small deletion) and four frequent polymorphisms. With MSSCP electrophoresis at a standard temperature profile (gel temperature 35-20-10 degrees C) we were able to detect 13 of 14 (93%) different nucleotide exchanges in 25 of 29 PCR fragments (86%). Hence, the detection rate for genetic variations in the FXI gene was 86%. To evaluate the reproducibility, MSSCP was performed twice for 174 PCR fragments and the consistency between two electrophoretic runs was 99%. We conclude that the MSSCP is a sensitive, fast, and cost effective screening method for the detection of FXI gene mutations.

Factor 11 single-nucleotide variants in women with heavy menstrual bleeding

“…Furthermore, rs2289252 was associated with miscarriages, decreased APTT and high FXI levels. (Ventura et al 2000;Zivelin et al 2002;Zadra et al 2004;Zadra et al 2008;Kim et al 2012;Bicocchi et al 2013) rs5970 c.1191T > C p.Gly397¼ Exon 11 VTE N (Gerdes et al 2004) N (Zivelin et al 2002;de Moerloose et al 2004;Zadra et al 2004;Bezak et al 2005;Jayandharan et al 2005;Zadra et al 2008;Kim et al 2012;Bicocchi et al 2013), (Martincic et al 1998) This SNV is located in intron 12 (c.1481-188) and the result of a C > T substitution. Eight other SNVs, including rs1593, rs3822057, rs925451, rs4253430, rs4253414, rs4253399, rs3733403 and rs3822056 also showed an association with venous thrombosis.…”

Novel application of the MSSCP method in biodiversity studies

“…For MSSCP analyses, three different temperatures are used to separate short DNA fragments, which vary in sequence but are of the same length, in non-denaturing polyacrylamide gel electrophoresis. Until now, MSSCP analyses have mostly been used for the detection of point mutations in specific genes [ 11 , 12 ] which are important in clinical medicine. Recently, Szewczyk et al .…”