Comparison of heteroduplex analysis, direct sequencing, and enzyme mismatch cleavage for detecting mutations in a large gene, FBN1.

Analysis of large genes for mutations of clinical relevance is complicated by intragenic heterogeneity, sensitivity, and cost of the methods available, and in the case of many conditions, specificity of the genetic alterations detected. We examined the FBN1 gene for mutations in people who had Marfan syndrome using three methods: single-chain polymorphism analysis (SSCP) with heteroduplex (HA) analysis, enzyme-mediated cleavage (EMC) of heteroduplexes, and direct sequencing. We also used these methods to search for mutations in the P53 gene in patients with hepatocellular carcinoma. The results showed that EMC was most efficient for detecting mutations. However, the cost favored SSCP with heteroduplex analysis, provided conditions did not need to be optimized to detect a mutation. Until more cost-effective and sensitive methods are developed to detect unknown mutations in large genes, diagnosis of many genetic disorders will depend on the willingness of an investigator who is studying a particular disorder to perform clinical molecular testing and have the laboratory accredited.