ANGIOTENSIN I-CONVERTING ENZYME (ACE) - ESTIMATION OF DNA HAPLOTYPES IN UNRELATED INDIVIDUALS USING DENATURING GRADIENT GEL BLOTS

The angiotensin I-converting enzyme (ACE) gene (17q23) is a candidate gene for essential hypertension and related diseases, but investigatio n of its role in human pathology is hampered by a lack of identified p olymorphisms. Currently, a 287-bp insertion/deletion (I/D) RFLP in int ron 16 represents the only one known. Additional polymorphisms for the ACE gene would make most families informative for linkage studies and would allow haplotypes to be assigned in association studies. To incr ease the information provided by the ACE gene, we used a sensitive scr eening technique, denaturing gradient gel electrophoresis (DGGE) blots , to identify polymorphisms and combined this with gene counting to id entify haplotypes. Five independent polymorphisms, restriction fragmen t melting polymorphisms (RFMPs), were identified by four probes (encom passing half of the ACE cDNA) in digests produced by three restriction enzymes (DdeI, RsaI, and AluI). One RFMP has three alleles while the others have two alleles. In a sample of 67 unrelated control subjects, minor allele frequencies ranged from 0.12 to 0.49. A significant leve l of linkage disequilibrium was found for all pairs of markers. The fo ur most informative RFMPs, taken in combination, define 24 potential h aplotypes. Based on gene counting, 11 of the 24 are rare or nonexisten t in this population, and the estimated heterozygosity of the remainin g 13 haplotypes approaches 80%. Under these conditions for the ACE loc us, phase-unknown genotypes could be assigned to haplotype pairs in un related subjects with reasonable certainty. Thus, using DGGE blot tech nique for identifying numerous DNA polymorphisms in a candidate locus, in combination with gene counting, one can often identify DNA haploty pes for both related and unrelated study subjects at a candidate locus . These markers in the ACE gene should be useful for clinical and epid emiologic studies of the role of ACE in human disease.