64 MACROGLOSSIA IN CLONED PIGLETS IS ASSOCIATED WITH HYPOMETHYLATION AT THE KCNQ-OT1 CpG ISLAND

Beckwith-Wiedeman Syndrome (BWS) is a loss of imprinting (LOI) condition that is associated with macroglossia, midline abdominal defects, and neonatal gigantism among other symptoms. These symptoms have also been seen in animals produced by SCNT. A common feature of BWS is the loss of methylation at the KCNQ-OT1 differentially methylated region. We hypothesised that this locus would show a similar LOI in cloned piglets that display macroglossia. DNA sequence for the porcine KCNQ-OT1 region was assembled in silico from public genome sequencing data. A CpG island was noted as being similarly located in the swine sequence as one which has been described for the human differentially differentiated region. Primers were designed to amplify a portion of this region from bisulfite converted genomic DNA. The amplimer spanned 32 CpG sites. To confirm imprinting status of KCNQ-OT1 in swine, a non-cloned pig was evaluated as to the methylation status across this region using DNA isolated from muscle (M) and the proportion hypermethylated was evaluated by chi-square tests. As seen in humans, this region was hypermethylated in approximately half (12 of 24, P = 1) of the cloned, sequenced amplimers. This observation is consistent with a parent of origin imprint at this locus. Next, 2 cloned piglets that appeared normal were assessed for methylation at KCNQ-OT1. M DNA from each of these animals was consistent with normal methylation at this locus, (7 of 16 and 8 of 18 cloned, sequenced amplimers, P > 0.40). Next, M DNA was isolated from 2 cloned litter mates where 1 piglet presented with macroglossia and the other did not. The non-presenting piglet’s M DNA was methylated in approximately half of the cloned sequenced amplimers (9 of 17, P = 0.67) whereas the macroglossia piglet M DNA was devoid of the methylated allele (0 of 14, P < 0.001). An additional pair of macroglossia presenting and non-presenting cloned littermates was identified. In this pair, the non-presenting piglet showed a normal distribution of methylation at this allele (8 of 19, P = 0.77) and the macroglossia piglet deviated somewhat from normal (6 of 20, P < 0.05). These 2 case studies are consistent with the conclusion that the appearance of macroglossia in cloned pigs may be associated with hypomethylation at KCNQ-OT1 and may model BSW. However, additional abnormal pigs will need to be assessed to completely characterise the LOI in cloned piglets.