Hardy-Weinberg disequilibrium and association study of insertion/deletion polymorphism of ACE gene and Alzheimer s disease in Egyptian patients

We thank Dr Saadat’s letter to the editor [1] for his critical remarks on our study.To clarify, Dr. Saadat raised concernswith respect to genotyping errors based on the observed deviation from Hardy–Weinberg equilibrium for this particular polymorphism in our trial. Genotyping error is indeed one of the many possible sources of Hardy–Weinberg disequilibrium. To exclude this possibility and because the D allele in heterozygous samples is preferentially amplified, as reported in our publication [2], each sample that found to have the DD genotype was subjected to a second, independent PCR amplification with a primer pair that recognizes an insertion-specific sequence, with identicalPCRconditionsexcept for an annealing temperature of 67 C. The reaction yields a 335-bp amplicon only in the presence of an I allele and no product in samples homozygous for DD. This procedure correctly identified sampleswith the DI genotype that aremisclassified as DD with the insertion-spanning primers, which we concern it as an insuring test of our results.