Genomic imprinting is an epigenetic modification that directs parent-specific gene expression. Imprinted genes are responsible for regulating growth and development of the conceptus . These genes are typically found in clusters containing both maternally- and paternally-expressed genes. The correct allelic expression of the clustered genes is regulated by a neighboring region of DNA which is differentially methylated and is known as the imprinting control region (ICR; [2–4]). The effect of the ICR on a cluster of imprinted genes can span for megabases in a bidirectional manner .
Imprinted genes are functionally haploid  and therefore are vulnerable to epigenetic mutations and loss-of-imprinting (LOI; ). LOI refers to the misregulation of imprinted gene expression which results in either loss of expression or biallelic expression of these genes.
There are several LOI disorders in humans including Beckwith-Wiedemann syndrome (BWS), Angelman syndrome, Prader-Willi syndrome, and Silver Russell syndrome. BWS is the most frequent LOI syndrome observed in humans with an incidence of one in 13,700 live births [8, 9]. BWS is also the most common pediatric overgrowth syndrome . The overgrowth parameters for height and weight for BWS patients are among the 97th percentile .
The primary features of BWS include macroglossia, macrosomia, and abdominal wall defects [10, 11]. The secondary features include visceromegaly, polyhydramnios, renal abnormalities, facial nevus flammeus, hypoglycemia, hemihyperplasia, ear creases and helical pits, and cardiac malformations [9–12]. Children with this syndrome also have an increased susceptibility (4–21%) to develop embryonic tumors by the time they turn five years of age [8, 13, 14]. Wilms’ tumor of the kidney is the most common embryonic tumor (67% of cases) observed in BWS patients .
BWS is thought to occur because of the dysregulation of several imprinted genes located primarily on chromosome 11p15.5 [9, 11, 15]. The two main imprinted gene clusters associated with BWS are those directed by the KvDMR1 and H19/IGF2 ICRs [12, 16]. The BWS-associated imprinted genes regulated by the KvDMR1 include the paternally-expressed non-coding RNA KCNQ1OT1 and the maternally expressed coding genes CDKN1C, KCNQ1, and PHLDA2. In mice, expression of CDKN1C is also regulated by a differentially-methylated region (DMR) of DNA that encompasses the promoter and extends through exon 2 [17, 18]. Contrary to what has been reported for mice, no differential methylation is observed for CDKN1C in humans .
The KvDMR1 is methylated on the maternal allele and unmethylated on the paternal allele in mouse and human. Loss of methylation (LOM) at the KvDMR1 on the maternal allele is the most common epigenetic defect (50%) observed in BWS patients [9, 12, 16, 20, 21]. This LOM results in the aberrant expression of the long noncoding RNA (ncRNA) KCNQ1OT1 from the maternal allele which results in bidirectional silencing of the maternally-expressed flanking genes, in particular CDKN1C[8, 22].
The H19/IGF2 ICR regulates the expression of the paternally-expressed gene IGF2 and the maternally-expressed ncRNA H19. This region is unmethylated on the maternal allele and methylated on the paternal allele . The gain of methylation on the maternal allele results in the repression of H19 from the maternal allele leading to biallelic expression of IGF2. This epimutation occurs in 2–10% of BWS patients and is highly associated with tumor development [9, 16, 23]. Recent studies have found that some BWS patients also have LOM at the HYMAI/PLAGL1, MEST, and GRB10 ICRs [24–26].
In humans PLAGL1 is found on chromosome six, unlike the other genes associated with BWS which are found primarily on chromosome 11. PLAGL1 functions as a tumor suppressor and can induce apoptosis [27, 28]. In a study by Arima et al., it was determined that PLAGL1 is expressed similarly to CDKN1C in many tissues. A recent microarray study  places PLAGL1 as a pivotal player in the regulation of expression of a network of imprinted genes, including H19, IGF2, and CDKN1C.
In ruminants there is an overgrowth syndrome that resembles BWS. The overgrowth syndrome in ruminants is known as large offspring syndrome (LOS; ). LOS has been documented to result from several embryo culture conditions [31–34] and high protein diet supplementation to the dam prior to conception and during early pregnancy . The phenotypical features of LOS include: increased birth weight, macrosomia, skeletal defects, hypoglycemia, polyhydramnios, visceromegaly, difficulty suckling, and perinatal death [30, 31, 36–38].
Currently, no animal models exist that recapitulate the overgrowth phenotype of BWS. Murine knockout models for BWS have been unable to display all the primary features observed in children with BWS . As an effort to develop treatments for BWS symptoms, our long-term goal is to determine if LOS in ruminants can be used as an animal model to understand the etiology of the LOI syndrome BWS. The goal of this paper was to ascertain baseline allelic expression and DNA methylation in control bovine concepti of imprinted genes/regions known to be misregulated in BWS. Similar to what has been previously reported [40, 41]; we show that KCNQ1OT1, H19, CDKN1C and PLAGL1 are imprinted in the bovine. In addition, we confirm that the KvDMR1 and H19/IGF2 ICR are differentially methylated in the bovine genome which is in accordance to what has been reported in humans. Our study extends previous work [40, 41] in that it provides fixed DNA sequence polymorphisms between Bos taurus indicus and Bos taurus taurus that can be used to distinguish with certainty the parental alleles in F1 individuals.