MISTYPING OF THE HUMAN ANGIOTENSIN-CONVERTING ENZYME GENE POLYMORPHISM - FREQUENCY, CAUSES AND POSSIBLE METHODS TO AVOID ERRORS IN TYPING

A polymorphism of the gene encoding the human angiotensin I-converting enzyme (ACE), which is defined by an insertion/deletion polymorphism in intron 16, has been identified as a candidate genetic locus in the development of cardiovascular and renal disease. We have demonstrated that the accuracy of ACE genotyping is critically dependent on the str ategy of the PCR used in typing. Of 1238 individuals genotyped by a st andard method, 335 were typed as DD, 645 as DI and 258 as II. However, when DD individuals were retyped using modified methods (including ei ther 5% dimethyl sulphoxide, or a 'hot start') 35 of the original 335 samples (10.5%) were retyped as DI. In approximately half of these mis typed samples, PCR amplification was assessed as inefficient by the ab sence of a third faint heteroduplex band in a control ID sample: when the assay was repeated without any modifications, the mistyped samples were correctly genotyped. In the remainder, mistyping persisted. In t hese cases, the use of a third 'nested' PCR primer specific for the I allele was required for successful genotyping, providing a more reliab le strategy without the need for further modification to the PCR techn ique. Our results suggest that the triple primer approach is the metho d of choice for accurate ACE genotyping.