Denaturing high-performance liquid chromatography for screening antithrombin III gene mutation and polymorphisms in patients with cerebral venous thrombosis / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To identify antithrombin III (AT-III) gene mutation and polymorphisms in pregnant women and parturients with cerebral venous thrombosis (CVT) using denaturing high-performance liquid chromatography (DHPLC).</p><p><b>METHODS</b>The genomic DNA was extracted from the blood samples of 50 pregnant women and parturients with CVT and 52 matched healthy women for molecular analysis using a PCR/DHPLC assay followed by DNA sequence analysis. Ten primer pairs were designed for amplifying the AT- III promoter region and exons 1-6 including the exon/intron boundaries. A rapid screening assay based on DHPLC was established to screen the mutation and polymorphisms of AT- III gene.</p><p><b>RESULTS</b>Six abnormal peaks were detected in 40 of the patients by DHPLC. Direct DNA sequencing was performed on representative samples detected by DHPLC profiling. One pathogenic heterozygous G13328A missense mutation in exon 6, and a novel silent mutation in exon 4+243 G>A were identified. Six single nucleotide polymorphism (SNP) sites were found, including 4 previously reported ones in the SNP library and two were novel SNP sites. An abnormal peak was detected in the control group by DHPLC.</p><p><b>CONCLUSION</b>DHPLC allows automated and rapid high-throughput detection of AT- III gene mutation and polymorphisms in the clinical setting and prenatal diagnosis. Our findings suggested that AT- III gene mutation, as well as its polymorphisms, contributes to the occurrence of CVT in pregnant women and parturients.</p>