Denaturing high-performance liquid chromatography technique platform applied to screen G6PD deficient variants.
OBJECTIVE
Of denaturing high performance liquid chromatography (DHPLC), a technique platform was developed for screening G6PD deficient variants.


METHODS
When applied to screen and identify the G6PD deficient variants from 124 patients who come from 11 nations in China, the DHPLC was compared with amplification refractory mutation system (ARMS) or DNA sequence technique and assessed carefully in its accuracy, sensitivity, efficiency and the cost of experiment.


RESULTS
The G6PD-deficient variants, such as 1388 G-->A (36/124 cases), 1376 G-->T(35), 95 A-->G (14), 1024 C-->T (3), 392 G-->T (4), 871 G-->A /1311 C-->T /IVS XI +93 t-->c (9), 871 G-->A (1), 1311 C-->T/IVS XI +93 t-->c (4), 1376 G-->T /1388 G-->A (1) and so on, were characterized as sharp peaks by DHPLC and verified by DNA sequence. Further, the standard chromatograms were put into database for 8 kinds of common G6PD deficient variants in Chinese populations. And also DHPLC found 3 G6PD variants (1388 G-->A) from 103 negative controls. With taking 8.8 minutes and costing 1 dollar for each sample, DHPLC successfully detected and identified 34 heterozygous females from patients with G6PD deficiency. However, ARMS checked 83 positive controls but got 12 false G6PD mutants, of which 5 were false positive, 7 false negative. Above results show that DHPLC sounds like to be more convenience, sensitive and accurate than ARMS and DNA sequence techniques for checking G6PD mutants.


CONCLUSIONS
DHPLC is of great advantage to screen the G6PD deficient variants with accuracy, convenience, automation and less cost, and significantly to identify the female heterozygote and clinical type IV individuals with G6PD deficiency.
Jiang
Wei-ying
WY
Medical Genetic Department, Basic Medical College, Sun Yat-sen University, Guangzhou, Guangdong, P. R. China. jweiying@yahoo.com
Chen
Lu-ming
LM
Lin
Qun-di
QD
Geng
Qian
Q
Du
Chuan-shu
CS
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004252:DNA Mutational Analysis; D005260:Female; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics*; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008297:Male; D009154:Mutation; D015203:Reproducibility of Results
D005954:Glucosephosphate Dehydrogenase