[Denaturing high-performance liquid chromatography for screening antithrombin III gene mutation and polymorphisms in patients with cerebral venous thrombosis].
OBJECTIVE
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wang
Li-ping
LP
Department of Gynecology and Obstetrics, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Qiu
Yu-wen
YW
Yin
Ai-lan
AL
Ma
Yun-yan
YY
Liu
Ke-ling
KL
Xiong
Li
L
Yu
Yan-hong
YH
Zhong
Mei
M
Wang
Chen
C
D000328:Adult; D000990:Antithrombin III / Q000235:genetics*; D001483:Base Sequence; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D020767:Intracranial Thrombosis / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011110:Polymorphism, Genetic / Q000235:genetics*; D011247:Pregnancy; D011250:Pregnancy Complications, Hematologic / Q000235:genetics*; D055815:Young Adult
D000990:Antithrombin III