BACKGROUND
genomic imprinting is the epigenetic phenomenon wherein genes are expressed exclusively from one parental allele  <cit> .
imprinting has been reported in placental mammals, specifically, in primates, rodents, canines and ruminants.
some of these imprinted genes exhibit species-specific and spatial-temporal patterns of imprinted expression  <cit> .
selective inactivation of one parental allele can be achieved by parent-of-origin specific cytosine methylation.
germline-derived heritable differentially methylated regions  are established at the gamete stage  <cit> .
secondary differentially methylated marks are acquired after fertilization or later in life, and these are known as somatic dmrs or sdmrs  <cit> .
allele-specific activating or repressive histone modifications have also been implicated in regulating imprinting  <cit> .
since the discovery of the first imprinted gene in  <dig>   <dig> imprinted genes have been identified in humans http://igc.otago.ac.nz/home.html while  <dig> imprinted genes have been reported in mice .
in recent years, many studies using genome-wide technologies for genomic or epigenomic analyses were performed to identify novel imprinted genes.
however, most had mixed success  <cit> .
whole genome and transcriptome sequencing technologies have helped identify only a small number of imprinted transcripts  <cit> , suggesting that most imprinted genes have already been identified  <cit>  or are tissue-specific and thus needed to be analyzed in specific cell types  <cit> .
only one study identified a large number of potential imprinted genes in the mouse brain  <cit> , but further investigation revealed that most of these may be false positives due to artifacts from the rna-seq approach  <cit> , a finding supported by more recent data  <cit> .
functionally, genomic imprinting is critical for proper placenta and embryo development  <cit> .
conditions such as intra-uterine growth restriction  and pre-eclampsia as well as unsuccessful pregnancies have been correlated with abnormalities in methylation or aberrant expression of imprinted genes in the placenta  <cit> .
surprisingly, very few human or primate-specific placental imprinted genes are known so far, though interesting candidates like rb <dig> , znf <dig> and the microrna cluster c19mc have been discovered in recent screens  <cit> .
a comparison between the  <dig> imprinted genes discovered to date in humans and the  <dig> reported in mice reveals that majority of this divergence is due to the multiple genes imprinted specifically in the mouse placenta  <cit> , although recent data suggests that several genes were wrongly identified as showing imprinted expression in mouse placenta  <cit> .
the imprinting difference is consistent with the biological differences between the less-invasive mouse placenta and its highly invasive human counterpart.
*based on ucsc genome build hg <dig>
**polymorphic imprinting reported in human placenta, but dmr still present.
in this study, we used reduced representation bisulfite sequencing  to identify partially methylated cpg islands  in the human placental genome.
we further identified candidate regions with allele-specific methylation based on calculation of methylation concordance values.
we then selected  <dig> regions for further characterization and identified two novel imprinted genes .
both genes are paternally expressed and methylated specifically on the maternal allele in the human placenta.
for aim <dig>  the differential methylation is conserved in another primate, the cynomolgus macaque but not in the mouse.
in conclusion, we have delineated many regions with allele-specific methylation and developed an approach for the identification of human placenta-specific imprinted genes.
RESULTS
confirmation of known germline differentially methylated regions using rrbs dna methylation analysis
nine human placental samples  were subject to rrbs analysis for dna methylation .
cpg sites sequenced at greater than 10× coverage were included in the analysis.
if our approach was to be used for identifying novel imprinted genes, it should also be able to confirm the known gdmrs.
indeed, cgis overlapping  <dig> known human dmrs  were found to be approximately 50% methylated .
the dmrs for the genes mcts <dig> and inpp5f_v <dig>  were further validated by bisulfite cloning and sequencing and were found to be methylated in an allele-specific manner .
the nnat promoter was not covered by our sequencing data.
for the gnas locus, the cgi overlapping with the dmr exhibited  <dig> % methylation.
however, on analyzing individual cpg sites within this large cgi, the first half of the cgi was found to be about 50% methylated.
allele-specific methylation analysis and selection of potential dmrs
on calculation of a concordance value , the known dmrs were shown to be partially methylated with high concordance .
the mean and median concordance values for the first trimester placentas were  <dig> % and  <dig> % respectively while those for the third trimester placentas were  <dig> % and  <dig> % respectively.
however, other partially methylated cgis  showed a much higher variability in concordance value.
we hypothesized that novel dmrs associated with imprinted genes should demonstrate similar methylation patterns to the known dmrs with partial methylation and high concordance.
by choosing partially methylated autosomal cgis with >85% concordance, we identified  <dig> regions in first trimester placenta and  <dig> regions in third trimester placenta,  <dig> of which were shared between the two trimesters .
these regions were located in promoters, gene bodies and inter-genic regions .
details of these regions are listed in additional file  <dig>
amongst the  <dig> potential dmrs overlapping between the first trimester and third trimester placenta samples, we chose  <dig> genomic regions for further validation.
the first  <dig> regions  were chosen based on high expression levels of adjacent genes in placenta as ascertained from rna-seq data .
the other  <dig> regions  were chosen since they were highly methylated in human spermatozoa samples  <cit>  and also had high expression levels of adjacent genes in placenta.
these  <dig> regions were candidates for paternally methylated gdmrs.
*based on ucsc genome build hg <dig>
*based on ucsc genome build hg <dig>
analysis of allele-specific expression for genes located in the selected regions in human placenta
we chose  <dig> genes  associated with the  <dig> candidate dmrs for analysis of allele-specific expression.
three to four exonic snps per gene were analyzed in  <dig> paired placental dna and rna samples.
two genes  showed allele-specific expression.
the monoallelic expression profile was not due to biased expression from one specific allele since reciprocal alleles were represented in the sample set .
for dnmt <dig> , eight heterozygotes  exhibited a single allele in their cdna .
for aim <dig> , there are two alternative transcripts, the long transcript and the short transcript .
allele-specific expression was observed in  <dig> individuals with two different snps located in exon  <dig> specific for the long transcript .
however, bi-allelic expression was observed with snps located in exon  <dig> shared by both the long and the short transcript in two individuals , indicating that imprinting is limited only to the long transcript of aim <dig>  the bi-allelic status of the shorter transcript was confirmed by 3’ race and snp analysis.
methylation and imprinting analyses of dnmt <dig> in human placenta
dnmt <dig> has two alternative transcripts, one expressed in somatic tissues  and the other expressed specifically in the oocyte .
the potential dmr identified at chromosome  <dig> cgi  <dig> is located at the promoter of s-dnmt <dig>  we performed bisulfite cloning and sequencing for this region in three human placenta samples to confirm the allele-specific methylation status .
an individual  with an informative snp exhibited methylation of the maternal allele  and was unmethylated on the paternal allele .
the allele-specific methylation profile was confirmed in one more sample .
however, parent-of-origin methylation could not be determined for the second sample since the mother was also heterozygous at this locus.
all the eight polymorphic human placenta samples showed monoallelic expression.
informative snps were available in four samples where the mothers were homozygous.
all four samples showed paternal allele-specific expression of s-dnmt <dig> .
methylation and imprinting analyses of aim <dig> in human placenta
we performed bisulfite cloning and sequencing for four human placental dna samples for the chromosome  <dig> cpg  <dig> region .
all four samples displayed equal numbers of methylated and unmethylated clones, characteristic of a dmr .
an individual  with an informative snp at the dmr exhibited a methylated maternal allele , while the paternal allele  was unmethylated .
this maternal allele-specific methylation profile was confirmed in one more individual .
we also confirmed that the methylation profile was not a snp effect by profiling a sample  that was non-polymorphic at the same locus.
this sample still showed the characteristic allele-specific methylation profile .
allele-specific expression was observed in  <dig> individuals with two different snps located in exon  <dig> specific for the long transcript.
four of the mothers were homozygous at the corresponding snp loci and thus were informative for parent-of-origin expression analysis.
all four placenta samples displayed paternal expression of this gene .
methylation and imprinting analyses of aim <dig> in cynomolgus macaque placenta
the region homologous to human cpg  <dig> in the macaque was analyzed in the placental dna of three macaques and shown to have approximately 34% methylation in each sample .
analysis of one macaque  with a c/g polymorphism within the dmr indicated that the expressed allele  was completely unmethylated, whereas the non-expressed allele  was partially methylated .
eleven macaque placental tissues were further analyzed for expression, and four heterozygotes for the first exon were found.
monoallelic expression was observed in all four samples .
since parent/offspring matched samples were not available, we were unable to determine the parental origin of the expressed allele in these animals.
methylation and imprinting analyses of aim <dig> in other cynomolgus macaque tissues
additional macaque tissues  were available for two of the informative individuals, and these were also subject to methylation analysis at the dmr.
however, all the tissues were found to be completely unmethylated .
aim <dig> was expressed in the heart, kidney and placenta, but showed minimal or no expression in the liver, lung and pancreas by qpcr analysis .
aim <dig> was found to be bi-allelically expressed in the kidney and heart in two macaques .
methylation and imprinting analyses of aim <dig> in mice
twelve samples from reciprocal crosses of cast/eij and bl <dig> mice were analyzed for methylation levels in the aim <dig> promoter, and in a second region with a potential alternative transcription start site upstream of aim <dig>  both regions were hypomethylated in this species .
aim <dig> was expressed in the kidney, placenta and heart but showed minimal expression in the liver, brain and lung  by qpcr analysis.
bi-allelic expression was observed in all tissues  in the reciprocal crosses .
discussion
the human placenta was chosen for our investigation of novel imprinted genes since genomic imprinting is critical for placenta and embryo development.
additionally, morphological and physiological differences are evident between mouse and human placenta, consistent with differences in imprinting between these two species.
rrbs was used to quantify dna methylation at cpg-rich regions, since it allowed us to readily distinguish two different types of partially methylated regions: those with allele-specific methylation which show high concordance; and those that exhibit variable methylation where different cpgs on the same allele can be methylated or unmethylated  <cit> .
our dna methylation data at single base resolution confirmed  <dig> known dmrs associated with imprinted genes.
one known dmr  was not confirmed because the genomic region was not analyzed by rrbs.
as expected, the known dmrs were partially methylated with high concordance.
thus, we selected  <dig> candidate dmrs from  <dig> partially methylated regions with high concordance in both first and third trimester placenta samples for analysis of allele-specific expression of adjacent genes.
subsequently, we confirmed that dnmt <dig> and aim <dig> were maternally methylated and paternally expressed.
while we were preparing the manuscript, a similar theoretical model was used to describe allele-specific methylation in the human genome  <cit> .
the authors identified known imprinted dmrs from publically available methylome datasets in predominantly cultured cells.
another related model has also been used to detect allele-specific methylation in the arabidopsis genome  <cit> .
the chromosome  <dig> dmr is located at the promoter of the well-studied dnmt <dig> gene.
dnmt <dig> selectively methylates hemi-methylated dna, regulates tissue-specific methylation and is also essential for maintenance of progenitor cells in an undifferentiated state in somatic tissues  <cit> .
it produces two transcripts, one expressed in somatic tissues  and the other expressed specifically in the oocyte .
the promoter of s-dnmt <dig> was shown to be monoallelically methylated specifically in the primate placenta and to be hypomethylated in other human tissues  <cit> .
novakovic et al.
reported the monoallelic methylation in human placenta to be random, based on only one sample harboring a snp  in the promoter of s-dnmt <dig>  in another recent report, an elegant screen for potential dmrs using diandric and digynic conceptuses also identified the same dmr at the s-dnmt <dig> locus, although again only one informative individual was analyzed to confirm monoallelic expression  <cit> .
in our study, we confirmed monoallelic expression in eight individual samples, paternal allele-specific expression in four of these individuals, and maternal specific methylation in one sample with an informative snp.
the latter snp is located in the cgi within the dnmt <dig> promoter .
thus, it is likely that placental genomic imprinting of dnmt <dig> is maintained throughout the primate lineage.
since we lacked parent-offspring matched samples for our macaque tissues, we were unable to confirm the parental-allele specific expression of dnmt <dig> in this species.
it is also interesting that the promoter of s-dnmt <dig> has been shown to be unmethylated in the mouse placenta .
however, dynamic methylation changes have been observed upstream of the o-dnmt <dig> transcript during early mouse development  <cit> .
no evidence for imprinting of murine dnmt <dig> has emerged from genome-wide placenta specific imprinting studies in mice  <cit> .
thus, it appears that genomic imprinting of dnmt <dig> is specific to the primate placenta.
the function of the paternal allele-specific expression of dnmt <dig> in human placenta remains to be elucidated.
methylation of the s-dnmt <dig> promoter may attenuate its transcription; this is coincident with global hypomethylation of the human placenta  <cit> .
moreover, s-dnmt <dig> expression attenuation has been reported to cause alterations in methylation at germline dmrs  <cit> .
thus, it is possible that reduction in s-dnmt <dig> level in the human placenta by genomic imprinting is linked to loss-of-imprinting observed at several loci in this tissue  <cit> .
aim <dig> or absent in melanoma  <dig> is a non-lens member of the βγ-crystallin superfamily  <cit> .
it was predicted to be a suppressor of malignant melanoma and nk-cell malignancies  <cit> .
it was implicated in trophoblast differentiation in the placenta  <cit> .
it has two alternative transcripts and both are highly expressed in the placenta.
the chromosome  <dig> dmr  lies at the exon1-intron <dig> junction of the long transcript of gene aim <dig>  460-bp downstream of the transcription start site.
there is another cpg island  located 587-bp upstream of the transcription start site of this transcript.
cpg  <dig> is unmethylated.
thus, cpg  <dig> potentially regulates the expression of the long transcript.
the dmr and imprinted expression were found to be conserved in the macaque placenta, but not in the mouse placenta.
it should be noted that in the macaque placenta the non-expressed allele was partially methylated while in the human placenta the non-expressed allele was fully methylated.
we suspect that this was due to maternal cell contamination in the macaque samples, since the macaque placenta is much thinner than the human placenta, making it difficult to isolate pure fetal cells.
it is interesting to speculate about the function of paternal allele-specific expression of the long transcript of aim <dig>  since this transcript appears to be robustly expressed in the placenta  <cit> , it is possible that its expression regulated by imprinting is functionally relevant in this tissue.
igf2r, one example of a maternally expressed imprinted gene is located on the same chromosome.
however, it is unlikely that they belong to the same imprinted cluster since they are approximately  <dig> mb apart.
moreover, igf2r exhibits polymorphic imprinting in humans  <cit> .
limited numbers of validated novel imprinted genes were discovered in previous genome-wide screens, raising the question whether most imprinted genes had been identified  <cit> .
despite evidence suggesting extensive loss of imprinting in the human placenta  <cit> , our study as well as others  <cit>  suggest that novel species and tissue-specific imprinted genes remain to be discovered  <cit> .
the functional consequences of such imprinting events may be species, tissue, and even developmental stage specific.
in this regard, the placenta may be a good tissue for studying genomic imprinting since it is both functionally important and evolutionarily under intense selective pressure.
however it is also clear from our data that while allele-specific dna methylation may be prevalent, most of these epigenetically regulated regions are not associated with genomic imprinting.
out of the  <dig> potential dmrs analyzed, only two were shown to be imprinted dmrs.
we confirmed the allele-specific methylation profile of  <dig> additional regions by bisulfite cloning and sequencing, even though these were not associated with monoallelic expression.
many of these potential dmrs are located in gene-bodies .
some of these regions could contribute to processes like alternative splicing  <cit> , or replication timing  <cit> .
one limitation of our study is the use of rrbs rather than whole genome bisulfite sequencing for the discovery of imprinted genes.
rrbs enriches for cpg-rich regions, particularly cpg islands.
it is possible that this led to identification of only maternally methylated dmrs in our study, since all known paternally methylated gdmrs are in cpg-poor, inter-genic regions.
analysis of the ten partially methylated regions which were fully methylated in sperm and were potential paternal gdmrs did not yield any imprinted gene candidates.
whole genome bisulfite sequencing analyses would facilitate future discovery of gdmrs in an unbiased manner.
CONCLUSIONS
in conclusion, we have developed a method to study allele-specific methylation and associated genomic imprinting in the human placenta.
careful follow-up and validation of other partially methylated regions with high concordance will potentially reveal the functional role of methylation in these regions and may help identify more novel imprinted genes.
the precise functions and mechanisms associated with placenta-specific imprinting of dnmt <dig> and aim <dig> remain to be investigated.
a more complete catalog of species-specific imprinted genes in the placenta will help our understanding of how genomic imprinting is associated with placental function, morphology and evolution.

 genomic imprinting is the epigenetic phenomenon wherein genes are expressed exclusively from one parental allele. imprinting has been reported in placental mammals, specifically, in primates, rodents, canines and ruminants. some of these imprinted genes exhibit species-specific and spatial-temporal patterns of imprinted expression. selective inactivation of one parental allele can be achieved by parent-of-origin specific cytosine methylation. germline-derived heritable differentially methylated regions are established at the gamete stage. secondary differentially methylated marks are acquired after fertilization or later in life, and these are known as somatic dmrs or sdmrs. allele-specific activating or repressive histone modifications have also been implicated in regulating imprinting. since the discovery of the first imprinted gene in imprinted genes have been identified in humans http://igc.otago.ac.nz/home.html while imprinted genes have been reported in mice. in recent years, many studies using genome-wide technologies for genomic or epigenomic analyses were performed to identify novel imprinted genes. however, most had mixed success. whole genome and transcriptome sequencing technologies have helped identify only a small number of imprinted transcripts, suggesting that most imprinted genes have already been identified or are tissue-specific and thus needed to be analyzed in specific cell types. only one study identified a large number of potential imprinted genes in the mouse brain, but further investigation revealed that most of these may be false positives due to artifacts from the rna-seq approach, a finding supported by more recent data. functionally, genomic imprinting is critical for proper placenta and embryo development. conditions such as intra-uterine growth restriction and pre-eclampsia as well as unsuccessful pregnancies have been correlated with abnormalities in methylation or aberrant expression of imprinted genes in the placenta. surprisingly, very few human or primate-specific placental imprinted genes are known so far, though interesting candidates like rb, znf and the microrna cluster c19mc have been discovered in recent screens. a comparison between the imprinted genes discovered to date in humans and the reported in mice reveals that majority of this divergence is due to the multiple genes imprinted specifically in the mouse placenta, although recent data suggests that several genes were wrongly identified as showing imprinted expression in mouse placenta. the imprinting difference is consistent with the biological differences between the less-invasive mouse placenta and its highly invasive human counterpart. *based on ucsc genome build hg **polymorphic imprinting reported in human placenta, but dmr still present. in this study, we used reduced representation bisulfite sequencing to identify partially methylated cpg islands in the human placental genome. we further identified candidate regions with allele-specific methylation based on calculation of methylation concordance values. we then selected regions for further characterization and identified two novel imprinted genes. both genes are paternally expressed and methylated specifically on the maternal allele in the human placenta. for aim the differential methylation is conserved in another primate, the cynomolgus macaque but not in the mouse. in conclusion, we have delineated many regions with allele-specific methylation and developed an approach for the identification of human placenta-specific imprinted genes.  confirmation of known germline differentially methylated regions using rrbs dna methylation analysis nine human placental samples were subject to rrbs analysis for dna methylation. cpg sites sequenced at greater than 10× coverage were included in the analysis. if our approach was to be used for identifying novel imprinted genes, it should also be able to confirm the known gdmrs. indeed, cgis overlapping known human dmrs were found to be approximately 50% methylated. the dmrs for the genes mcts and inpp5f_v were further validated by bisulfite cloning and sequencing and were found to be methylated in an allele-specific manner. the nnat promoter was not covered by our sequencing data. for the gnas locus, the cgi overlapping with the dmr exhibited % methylation. however, on analyzing individual cpg sites within this large cgi, the first half of the cgi was found to be about 50% methylated. allele-specific methylation analysis and selection of potential dmrs on calculation of a concordance value, the known dmrs were shown to be partially methylated with high concordance. the mean and median concordance values for the first trimester placentas were % and % respectively while those for the third trimester placentas were % and % respectively. however, other partially methylated cgis showed a much higher variability in concordance value. we hypothesized that novel dmrs associated with imprinted genes should demonstrate similar methylation patterns to the known dmrs with partial methylation and high concordance. by choosing partially methylated autosomal cgis with >85% concordance, we identified regions in first trimester placenta and regions in third trimester placenta, of which were shared between the two trimesters. these regions were located in promoters, gene bodies and inter-genic regions. details of these regions are listed in additional file amongst the potential dmrs overlapping between the first trimester and third trimester placenta samples, we chose genomic regions for further validation. the first regions were chosen based on high expression levels of adjacent genes in placenta as ascertained from rna-seq data. the other regions were chosen since they were highly methylated in human spermatozoa samples and also had high expression levels of adjacent genes in placenta. these regions were candidates for paternally methylated gdmrs. *based on ucsc genome build hg *based on ucsc genome build hg analysis of allele-specific expression for genes located in the selected regions in human placenta we chose genes associated with the candidate dmrs for analysis of allele-specific expression. three to four exonic snps per gene were analyzed in paired placental dna and rna samples. two genes showed allele-specific expression. the monoallelic expression profile was not due to biased expression from one specific allele since reciprocal alleles were represented in the sample set. for dnmt, eight heterozygotes exhibited a single allele in their cdna. for aim, there are two alternative transcripts, the long transcript and the short transcript. allele-specific expression was observed in individuals with two different snps located in exon specific for the long transcript. however, bi-allelic expression was observed with snps located in exon shared by both the long and the short transcript in two individuals, indicating that imprinting is limited only to the long transcript of aim the bi-allelic status of the shorter transcript was confirmed by 3’ race and snp analysis. methylation and imprinting analyses of dnmt in human placenta dnmt has two alternative transcripts, one expressed in somatic tissues and the other expressed specifically in the oocyte. the potential dmr identified at chromosome cgi is located at the promoter of s-dnmt we performed bisulfite cloning and sequencing for this region in three human placenta samples to confirm the allele-specific methylation status. an individual with an informative snp exhibited methylation of the maternal allele and was unmethylated on the paternal allele. the allele-specific methylation profile was confirmed in one more sample. however, parent-of-origin methylation could not be determined for the second sample since the mother was also heterozygous at this locus. all the eight polymorphic human placenta samples showed monoallelic expression. informative snps were available in four samples where the mothers were homozygous. all four samples showed paternal allele-specific expression of s-dnmt. methylation and imprinting analyses of aim in human placenta we performed bisulfite cloning and sequencing for four human placental dna samples for the chromosome cpg region. all four samples displayed equal numbers of methylated and unmethylated clones, characteristic of a dmr. an individual with an informative snp at the dmr exhibited a methylated maternal allele, while the paternal allele was unmethylated. this maternal allele-specific methylation profile was confirmed in one more individual. we also confirmed that the methylation profile was not a snp effect by profiling a sample that was non-polymorphic at the same locus. this sample still showed the characteristic allele-specific methylation profile. allele-specific expression was observed in individuals with two different snps located in exon specific for the long transcript. four of the mothers were homozygous at the corresponding snp loci and thus were informative for parent-of-origin expression analysis. all four placenta samples displayed paternal expression of this gene. methylation and imprinting analyses of aim in cynomolgus macaque placenta the region homologous to human cpg in the macaque was analyzed in the placental dna of three macaques and shown to have approximately 34% methylation in each sample. analysis of one macaque with a c/g polymorphism within the dmr indicated that the expressed allele was completely unmethylated, whereas the non-expressed allele was partially methylated. eleven macaque placental tissues were further analyzed for expression, and four heterozygotes for the first exon were found. monoallelic expression was observed in all four samples. since parent/offspring matched samples were not available, we were unable to determine the parental origin of the expressed allele in these animals. methylation and imprinting analyses of aim in other cynomolgus macaque tissues additional macaque tissues were available for two of the informative individuals, and these were also subject to methylation analysis at the dmr. however, all the tissues were found to be completely unmethylated. aim was expressed in the heart, kidney and placenta, but showed minimal or no expression in the liver, lung and pancreas by qpcr analysis. aim was found to be bi-allelically expressed in the kidney and heart in two macaques. methylation and imprinting analyses of aim in mice twelve samples from reciprocal crosses of cast/eij and bl mice were analyzed for methylation levels in the aim promoter, and in a second region with a potential alternative transcription start site upstream of aim both regions were hypomethylated in this species. aim was expressed in the kidney, placenta and heart but showed minimal expression in the liver, brain and lung by qpcr analysis. bi-allelic expression was observed in all tissues in the reciprocal crosses. discussion the human placenta was chosen for our investigation of novel imprinted genes since genomic imprinting is critical for placenta and embryo development. additionally, morphological and physiological differences are evident between mouse and human placenta, consistent with differences in imprinting between these two species. rrbs was used to quantify dna methylation at cpg-rich regions, since it allowed us to readily distinguish two different types of partially methylated regions: those with allele-specific methylation which show high concordance; and those that exhibit variable methylation where different cpgs on the same allele can be methylated or unmethylated. our dna methylation data at single base resolution confirmed known dmrs associated with imprinted genes. one known dmr was not confirmed because the genomic region was not analyzed by rrbs. as expected, the known dmrs were partially methylated with high concordance. thus, we selected candidate dmrs from partially methylated regions with high concordance in both first and third trimester placenta samples for analysis of allele-specific expression of adjacent genes. subsequently, we confirmed that dnmt and aim were maternally methylated and paternally expressed. while we were preparing the manuscript, a similar theoretical model was used to describe allele-specific methylation in the human genome. the authors identified known imprinted dmrs from publically available methylome datasets in predominantly cultured cells. another related model has also been used to detect allele-specific methylation in the arabidopsis genome. the chromosome dmr is located at the promoter of the well-studied dnmt gene. dnmt selectively methylates hemi-methylated dna, regulates tissue-specific methylation and is also essential for maintenance of progenitor cells in an undifferentiated state in somatic tissues. it produces two transcripts, one expressed in somatic tissues and the other expressed specifically in the oocyte. the promoter of s-dnmt was shown to be monoallelically methylated specifically in the primate placenta and to be hypomethylated in other human tissues. novakovic et al. reported the monoallelic methylation in human placenta to be random, based on only one sample harboring a snp in the promoter of s-dnmt in another recent report, an elegant screen for potential dmrs using diandric and digynic conceptuses also identified the same dmr at the s-dnmt locus, although again only one informative individual was analyzed to confirm monoallelic expression. in our study, we confirmed monoallelic expression in eight individual samples, paternal allele-specific expression in four of these individuals, and maternal specific methylation in one sample with an informative snp. the latter snp is located in the cgi within the dnmt promoter. thus, it is likely that placental genomic imprinting of dnmt is maintained throughout the primate lineage. since we lacked parent-offspring matched samples for our macaque tissues, we were unable to confirm the parental-allele specific expression of dnmt in this species. it is also interesting that the promoter of s-dnmt has been shown to be unmethylated in the mouse placenta. however, dynamic methylation changes have been observed upstream of the o-dnmt transcript during early mouse development. no evidence for imprinting of murine dnmt has emerged from genome-wide placenta specific imprinting studies in mice. thus, it appears that genomic imprinting of dnmt is specific to the primate placenta. the function of the paternal allele-specific expression of dnmt in human placenta remains to be elucidated. methylation of the s-dnmt promoter may attenuate its transcription; this is coincident with global hypomethylation of the human placenta. moreover, s-dnmt expression attenuation has been reported to cause alterations in methylation at germline dmrs. thus, it is possible that reduction in s-dnmt level in the human placenta by genomic imprinting is linked to loss-of-imprinting observed at several loci in this tissue. aim or absent in melanoma is a non-lens member of the βγ-crystallin superfamily. it was predicted to be a suppressor of malignant melanoma and nk-cell malignancies. it was implicated in trophoblast differentiation in the placenta. it has two alternative transcripts and both are highly expressed in the placenta. the chromosome dmr lies at the exon1-intron junction of the long transcript of gene aim 460-bp downstream of the transcription start site. there is another cpg island located 587-bp upstream of the transcription start site of this transcript. cpg is unmethylated. thus, cpg potentially regulates the expression of the long transcript. the dmr and imprinted expression were found to be conserved in the macaque placenta, but not in the mouse placenta. it should be noted that in the macaque placenta the non-expressed allele was partially methylated while in the human placenta the non-expressed allele was fully methylated. we suspect that this was due to maternal cell contamination in the macaque samples, since the macaque placenta is much thinner than the human placenta, making it difficult to isolate pure fetal cells. it is interesting to speculate about the function of paternal allele-specific expression of the long transcript of aim since this transcript appears to be robustly expressed in the placenta, it is possible that its expression regulated by imprinting is functionally relevant in this tissue. igf2r, one example of a maternally expressed imprinted gene is located on the same chromosome. however, it is unlikely that they belong to the same imprinted cluster since they are approximately mb apart. moreover, igf2r exhibits polymorphic imprinting in humans. limited numbers of validated novel imprinted genes were discovered in previous genome-wide screens, raising the question whether most imprinted genes had been identified. despite evidence suggesting extensive loss of imprinting in the human placenta, our study as well as others suggest that novel species and tissue-specific imprinted genes remain to be discovered. the functional consequences of such imprinting events may be species, tissue, and even developmental stage specific. in this regard, the placenta may be a good tissue for studying genomic imprinting since it is both functionally important and evolutionarily under intense selective pressure. however it is also clear from our data that while allele-specific dna methylation may be prevalent, most of these epigenetically regulated regions are not associated with genomic imprinting. out of the potential dmrs analyzed, only two were shown to be imprinted dmrs. we confirmed the allele-specific methylation profile of additional regions by bisulfite cloning and sequencing, even though these were not associated with monoallelic expression. many of these potential dmrs are located in gene-bodies. some of these regions could contribute to processes like alternative splicing, or replication timing. one limitation of our study is the use of rrbs rather than whole genome bisulfite sequencing for the discovery of imprinted genes. rrbs enriches for cpg-rich regions, particularly cpg islands. it is possible that this led to identification of only maternally methylated dmrs in our study, since all known paternally methylated gdmrs are in cpg-poor, inter-genic regions. analysis of the ten partially methylated regions which were fully methylated in sperm and were potential paternal gdmrs did not yield any imprinted gene candidates. whole genome bisulfite sequencing analyses would facilitate future discovery of gdmrs in an unbiased manner.  in conclusion, we have developed a method to study allele-specific methylation and associated genomic imprinting in the human placenta. careful follow-up and validation of other partially methylated regions with high concordance will potentially reveal the functional role of methylation in these regions and may help identify more novel imprinted genes. the precise functions and mechanisms associated with placenta-specific imprinting of dnmt and aim remain to be investigated. a more complete catalog of species-specific imprinted genes in the placenta will help our understanding of how genomic imprinting is associated with placental function, morphology and evolution. 
such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. we describe here a new strategy to study allele-specific dna methylation in the human placenta for the discovery of novel imprinted genes. imprinted expression for aim was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse. both genes showed maternal allele-specific methylation and paternal allele-specific transcription. our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions.
 genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. this is particularly relevant for the placenta. study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. we describe here a new strategy to study allele-specific dna methylation in the human placenta for the discovery of novel imprinted genes.  using this methodology, we confirmed differentially methylated regions associated with known imprinted genes. we chose genomic regions for further testing and identified two imprinted genes. both genes showed maternal allele-specific methylation and paternal allele-specific transcription. imprinted expression for aim was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse.  our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. their identification may help us better understand embryonic and fetal development. genomic imprintingplacentanext generation sequencingdifferentially methylated region dnmt1aim1 