Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in multiple tissues and organs. TSC exhibits locus heterogeneity with genes at 9q34 (TSC1) and 16p13.3 (TSC2) that have 21 and 41 coding exons, respectively. The mutational spectrum at both loci is wide and previous studies have shown that 60%-70% of cases are sporadic and represent new mutations. We have formatted denaturing high performance liquid chromatography (DHPLC) for rapid screening of all coding exons of T SC1 and TSC2. DHPLC analysis detected likely disease-causing mutations in 1 03 of 150 unrelated cases (68%), compared with 92/150 (61%) and 87/150 (58% ) fdr single-strand conformation polymorphism analysis (SSCP) and conventio nal heteroduplex analysis (HA), respectively. Capital, consumable and labou r costs were determined for each exon screening procedure. Estimated costs per patient sample depended on throughput, particularly for DHPLC, where a high proportion of costs are fixed, and were pound 257, pound 216 and pound 242 for DHPLC, SSCP and HA, respectively, assuming a throughput of 252 sam ples per year, or pound 354, pound 233 and pound 259, assuming a throughput of 126 samples per year. DHPLC had the advantages of increased sensitivity and reduced labour costs when compared with more traditional approaches to exon screening but, unless expensive DHPLC equipment is being efficiently utilised for a very high proportion of the time available, overall costs ar e slightly higher.