Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria: comparison with denaturing gradient gel electrophoresis

Acute intermittent porphyria is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding fo r porphobilinogen deaminase (PBGD). Many different strategies have been dev eloped to screen for mutations. However the high prevalence (0.6 parts per thousand) of PBGD gene defect, the large allelic heterogeneity of mutations (n = 130), and the limitations of the PBGD enzymatic assay for asymptomati c patients' detection, require for diagnosis an efficient and easy to handl e strategy for locating mutations within the PBGD gene. In a recent study t he sensitivity of the denaturing gradient gel electrophoresis (DGGE) techni que was 100%. However DGGE requires the preparation of gradient gels and th e use of primers with long GC-clamps; thus alternative methods should be pr eferable in the clinical laboratory. We have compared the detection rate of DGGE with heteroduplex analysis (HA) using 16 characterized PBGD gene muta tions. Six different HA conditions were used to determine the efficiency of the method, including: (1) MDE(TM) (mutation detection enhancement) gel co ncentration; (2) addition of urea and sodium dodecyl sulfate (SDS); (3) rad ioactive labelling. The sensitivity of each HA condition varied from 31 to 81% vs. 100% in DGGE analysis. HA using 1 X MDE(TM) With 15% urea with or w ithout 0.55% SDS was the most sensitive condition. This first comparative s tudy of DGGE and HA mutation screening methods suggests that DGGE is a more sensitive screening assay than optimized HA. However, because of its simpl icity HA should be considered as an efficient alternative mutation screenin g method. (C) 1999 Elsevier Science B.V. All rights reserved.