27 HIGHLY VARIABLE EPIGENOMES IN HEALTHY ADULT BOVINE CLONES

Abstract

Somatic cell nuclear transfer (SCNT) cloning requires epigenetic reprogramming of a differentiated donor cell nucleus. Incorrect reprogramming of epigenetic marks such as methylation of cytosine (5 mC) is associated with compromised development. Clones that survive into adulthood, in contrast, are assumed to be normal and are expected to be used in global food production. However, the epigenetic status of such healthy adult clones has never been investigated. We used a capillary electrophoresis technique to measure 5 mC content in leukocyte DNA from 38 healthy female clones at 1 to 8 years of age. Fibroblasts from 9 adults were used to generate 2 to 9 clones per donor. Four of the studied clone genotypes were from the Holstein breed, and 5 were from the Simmental breed. Individual 5 mC levels ranged from 4.4 to 6.9% with significant differences in mean 5 mC levels of Holstein and Simmental clones (6.50 ± 0.01% and 5.09 ± 0.02%, P < 0.001). The observed variances in 5 mC between clone genotypes of both breeds were similar (0.0204 and 0.0164), and we computed exact restricted likelihood ratio tests on the basis of linear mixed effects models to test for the presence of variability between genotypes. We found that the estimated variance in 5 mC level within clone genotypes from both breeds (0.104) was higher than the estimated variance between clone genotypes (<10–10). Three Holstein donors were analyzed for the 5 mC level of their genome together with their clones. Two donors exhibited the lowest 5 mC levels of the respective genotypes. The 5 mC level of the third nuclear donor was identical to 1 of its 8 clones, higher than 5 of them, and lower than the 2 remaining ones. This clearly demonstrates that the functional reprogramming of a given donor genotype is compatible with a highly flexible methylation status of its DNA and that genomic copies of adult animals have to be considered as epigenome variants. To quantify the contribution of SCNT to the observed variability between clones, we compared Simmental clones with female Simmental monozygotic twins of similar age that were generated by bisection of fertilized embryos. The estimated variability of 5 mC levels within 5 genotypes of clones (0.0636, n = 19) was clearly higher than in 12 twin pairs (0.0091). In clones, the estimated variability within genotypes (0.0636) was thus higher than between (<10–10) genotypes. In contrast, the variability within twin genotypes (0.0091) was lower than between twin genotypes (0.0136). Absolute deviations of 5 mC values of SCNT clones from their genotype means were 5-fold increased as compared with twins, whereas deviations of genotype means from group means were similar for clones and twins. The comparison with twins confirmed a SCNT cloning effect illustrated by a clone-specific variability of DNA methylation and provided new evidence of DNA hypermethylation by SCNT. Our data show that SCNT cloning can no longer be considered (epi)genetically neutral in apparently healthy adult animals. This raises new scientific and regulatory questions about the risks of disseminating cloned (epi)genetics into animal populations. Supported by EU (SABRE), DFG, INRA, and MRES.