DHPLC-based germline mutation screening in the analysis of the VHL tumor suppressor gene: usefulness and limitations

In order to evaluate the sensitivity and specificity of the recently introd uced high-throughput method DHPLC (denaturing high performance liquid chrom atography) for mutation screening in the VHL tumor suppressor gene, we subj ected DNA from 43 unrelated VHL patients with previously sequenced VHL germ line mutations to this method. In addition, 36 genomic DNAs of unrelated in dividuals suspected of being VHL carriers but with unknown germline status were analyzed by DHPLC and sequencing. The aims of the present study were t o compare mutation results obtained by direct sequencing and DHPLC, and a c omparison of two different DHPLC systems. The sensitivity of DHPLC was test ed with two commercial devices and protocols, i.e., the Varian-Helix system and the Wave Nucleic Acid Fragment Analysis system. Both resolved all but one mutation in exons 2 and 3 of the VHL gene. In contrast, the CC-rich exo n 1 showed discrepancies in the rate of mutation detection. Whereas the Var ian-Helix system detected 10/15 (67%) of the known mutations, the Wave Nucl eic Acid Fragment analysis system detected 13/14 (93%). All three mutations in samples with unknown mutation status were revealed by both systems rais ing the mutation detection rate to 72% and 94%, respectively. Cases with di fferent substitutions at the same nucleotide showed different elution profi les, but similar elution profiles could be obtained from different mutation s. The Wave Nucleic Acid Fragment Analysis system detected most VHL mutatio ns; however, when a 100% detection rate is needed, sequencing is still requ ired and must therefore be the standard VHL mutation detection procedure. O nce a family-specific mutation has been established, DHPLC may be suitable for the rapid and cost-effective determination of VHL carrier status in fam ily members.