Author Response: Nonspecific PCR Amplification of CRYBB2-Pseudogene Leads to Misconception of Natural Variation as Mutation

We appreciate the experiments and in silico studies of Kumar and Santhiya calling attention to the importance of addressing the risk of nonspecific amplification of the CRYBB2 pseudogene, CRYBB2-P1. ¹ Likewise, we acknowledge their notice on errors in the list of primer pairs published as Supplementary Table S1 in our report on mutations in Danish families with congenital cataract. ²  

We repeated the amplification of exon 5 of CRYBB2 in one individual from family CC00133 with the claimed mutations due to gene conversion, in one affected individual from each of four additional families with congenital cataract, and in one healthy individual. Amplifications were performed with the primer pair used originally for the analyses of the family, as well as the two primer pairs discussed in the letter by Kumar and Santhiya, ¹ Weisschuh et al., ³ and Santhiya et al. ⁴ The original PCR primer pair designed for amplification of CRYBB2 exon5 was designed so that the forward primer (ex5f-GTGTGCAAGTGTGGTGTGC) could not anneal to the pseudogene, CRYBB2-P1, whereas the reverse primer (ex5r-GAAGCCAGAGGTCAGCAGAG) was unable to discriminate between the two genes. Only family CC00133 of the 12 CC-families investigated at that time showed the three DNA variations in cis. We were, however, unable to reproduce the results from the original study and our results were now in complete accordance with the results of Kumar and Santhiya. Unfortunately we have no explanation for the erroneous results obtained in the original study. ² In conclusion, the three variations claimed to be located in cis in exon 5 of CRYBB2 in family CC00133 are, in fact, located in the pseudogene, CRYBB2-P1, and the postulated gene conversion erroneous. The mutation will be redrawn in a separate erratum in addition to a corrected Table S1. We thank Kumar and Santhiya for bringing up the discussion.