since it has been reported to have secondary effects on CHRNA5 mRNA levels37. The goal was to represent isolated genotypes in order to test the difference between the major and minor allele of rs16969968. It has been suggested that cell lines from multiple patients and controls as well as multiple subclones from each patient should be analyzed to provide a convincing cellular or functional phenotype. However, the resulting high workload is a major obstacle to the identification of disease-associated phenotypes434445. The most convincing solution to this problem is the creation of isogenic pairs of cells differing only in a single locus, usually by genetic engineering techniques4647. Indeed, our previous RNAseq analyses demonstrate broad variability among subject iPSCs, even among members of the same family, and a vastly reduced variability when comparing isogenic samples42. However, editing to create an isogenic rs16969968 iPSC is time consuming and technically challenging. Nevertheless, our results derived from different subjects and different subtypes of human neuronal population are consistent and possibly more relevant overall to human populations. Therefore, we believe that by testing a reasonable number of subject-specific iPSC lines we capture sufficient variability while searching for reproducible phenotypes based on the non-synonymous SNP.
