genomic imprinting is an epigenetic phenomenon that causes a differential expression of paternally and maternally inherited alleles of a minor subset of genes ( the so - called imprinted genes ) . genomic imprinting was first discovered in 1984 [ 1 , 2 ] , and in 1991 the first imprinted genes ( igf2 , paternally expressed ; igf2r and h19 , maternally expressed ) were identified in the mouse [ 35 ] . since then , the imprinting status was confirmed for numerous genes in homo sapiens and mus musculus genomes , less for bos taurus , rattus norvegicus , sus scrofa , canis lupus familiaris , and ovis aries ; many more genes are considered candidates . functional significance of the genomic imprinting is not yet fully understood [ 79 ] , while alterations in the expression of imprinted genes are linked to certain pathologies , including angelman syndrome , prader - willi syndrome , and particular cancer subtypes . furthermore , it is a dynamic process and may vary depending on the developmental stage . the goal of the study was to estimate a prevalence of imprinted genes within the total human transcriptome , in cell and tissue samples in a variety of physiological and pathological conditions . serial analysis of gene expression ( sage ) is a sequence - based technique to study mrna transcripts quantitatively in cell populations . two major principles underline sage : first , short ( 10  bp ) expressed sequenced tags ( ests ) are sufficient to identify individual gene products , and second , multiple tags can be concatenated and identified by sequence analysis . sage results are reported in either absolute or relative numbers of tags , which permits direct comparisons between tag catalogues and datasets [ 1215 ] . numerous technical adaptations assured a development of similar techniques , yet sage remains an important tool of modern molecular biology . it is widely used in a number of applications , of which a molecular dissection of cancer genome is the major . in the current study , expression of established and candidate imprinted genes was evaluated in a wide array of cell and tissue samples using a comprehensive set of currently available sage data for homo sapiens . five hundred eighty - one sage catalogues based on the libraries generated with most commonly used nlaiii anchoring enzyme were screened using a conservative set of criteria , and in 492 of these ( accounting for nearly 36 million sage tags ) gene expression profiles of the imprinted genes were analyzed , using a proved algorithm . it was therefore possible to estimate a prevalence of imprinted genes within the total human transcriptome . established and candidate imprinted gene subset was assembled based on the geneimprint resource ( http://www.geneimprint.com/ ; credits to r.l . high - confidence imprinted human gene candidates predicted to be imprinted by both the linear and rbf kernel classifiers learned by equbits foresight and by smlr ( , supplementary data ) were utilized . sage technology is based on isolation of short tags form the appropriate position within the mrna molecule , followed by the concatemerization of the tags , sequencing , tag extraction and gene annotation . the complete set of publicly available sage libraries ( gpl4 dataset , nlaiii anchoring enzyme ) was downloaded from the gene expression omnibus ( geo ) database ( national center of biotechnology information ( ncbi ) ; http://www.ncbi.nlm.nih.gov/geo/ ) . following an exclusion of the duplicate entries , sage libraries were annotated and sorted based on the number of tags sequenced . noninformative ( a)10 sequences were extracted from sage libraries when detected , and tags per million ( tpm ) values were recalculated accordingly for all libraries as the transcript 's raw tag count divided by the number of reliable tags in the library and multiplied by 1,000,000 . sage libraries , constructed by potapova et al . , were a subject to a clean - up  procedure through which all clones containing 4 tags were excluded , with the remaining tags constituting the pool of  reliable tags .  established and candidate imprinted gene subset has matched cgap ( cancer genome anatomy project , nci , nih ) sage anatomic viewer ( sav ) applet . for genes not matching sav applet entries , and when unreliable / internal tags were suggested by sav applet ( viz . , for tigd1 , hoxa3 , ntri genes , etc . ) , reliable 3 end tags were extracted from full - length sequences available via genbank ( ncbi , nih ) . sage tags was matched the individual sage catalogues using ms access software package query function . individual queries ( both absolute tag abundance per library and normalized tag per million ( tpm ) values ) were merged using ms excel software . calculations of maximal and average expression of transcripts matching established and candidate imprinted genes were performed using normalized tpm values . particular values could be recalculated to the fraction of the total gene expression by dividing tpm value by 1,000,000 . clustering analysis was performed using epclust expression profile data clustering and analysis software ( http://www.bioinf.ebc.ee/ep/ep/epclust/ ) . k - mean clustering analysis was performed after transposing the data matrix with initial clusters chosen by most distant ( average ) transcripts . for each dataset , the number of clusters was set to the lowest value yielding one cluster containing a solitary database entry . hierarchical clustering was performed using correlation measure - based distance / average linkage ( average distance ) clustering method ; hierarchical trees were built for individual datasets . established and candidate human imprinted gene subset ( 203 entries total ) was assembled based on the geneimprint resource and luedi et al . study data . of the candidate imprinted genes identified in the latter , high - confidence gene candidates ( predicted via equbits foresight and smlr means ) were selected . following exclusion of the redundant entries , appropriate short ( 10  bp ) sage tags matching nlaiii anchoring enzyme were annotated to gene targets using cgap ( cancer genome anatomy project , nci , nih ) sage anatomic viewer ( sav ) applet or manually , as described earlier . for a number of the candidate imprinted genes , a complete sequence was unavailable via genbank or alternative databases ( e.g. , genbank i d : nm_016158 , nm_024547 , nm_181648 , etc . ) , for that reason , a volume of the human imprinted gene subset subjected to tag annotation was reduced to 174 genes . of these genes , candidate imprinted gene q9nyi9 ( pparl ; genbank i d : af242527 ) could not be annotated with sage tag , missing nlaiii anchoring enzyme recognition sites completely . therefore , a total of 173 genes ( including 53 established imprinted genes and 120 candidate imprinted genes ) were annotated with the appropriate sage tags ( table 1 ) and subjected to further analysis . the complete set of publicly available human sage catalogues was downloaded from the gene expression omnibus ( geo , ncbi ) database . acquired sage catalogues represent 581 sage libraries generated from a wide spectrum of cell and tissue samples in a variety of physiological and pathological conditions . following an exclusion of the numerous duplicate geo database entries ( e.g. , gsm785 = gsm383907 , the criteria listed below were applied when selecting libraries for the analysis of gene expression . sage libraries were selected only if they have represented ( i ) genetically unaltered / unmodified samples , ( ii ) sage catalogues with a total number of tags 20,000 , and ( iii ) a complete dataset available . for example , samples gsm383929 and gsm180669 were excluded since these did not satisfy criteria ( i ) , representing ovary surface epithelium immortalized with sv40 and lymphocytes from down syndrome children , respectively ; samples gsm384024 ( white blood cells , cd45 , isolated from a mammary gland carcinoma ; 18,741 tags ) and gsm1128 ( breast cancer cell line ; tags detected once are not available ) were excluded as not satisfying criteria(ii ) and ( iii ) , respectively ( supplementary table 1 ) . due to the conservative nature of the criteria listed above , a total number of sage catalogues satisfying these and thus selected for further analysis ( i.e. , to the extraction of tags matching imprinted genes ) was reduced to 492 . together , these 492 sage catalogues representing human samples account for 35.97 million sage tags constructed using nlaiii - anchoring enzyme . the catalogues were assigned into one of the following clusters : c ( cancer tissue ; 185 sage catalogues ) , n ( normal tissue and cells ; 166 sage catalogues ) , iv ( cells cultured in vitro ; 112 sage catalogues ) , or d ( nontumorous disease tissue and cells ; 29 sage catalogues ) ( table 2 , and supplementary table 1 ) . figure 1 shows a distribution of the analyzed established and candidate imprinted genes through the human genome . primary analysis of the normalized expression profiles of the imprinted genes demonstrated a great variability in the cumulative gene expression for 173 genes ( figure 2 , table 3 , and supplementary table 2 ) . average cumulative gene expression of those genes in human tissues and cells was 0.90% of the total gene expression : specifically , 0.95% for both cancer and normal tissue and cells ( clusters c and n , resp . ) , 0.77% for cells cultured in vitro ( cluster iv ) , and 0.83% for nontumorous disease tissue and cells ( cluster d ) . in the pool of the assessed sage catalogues , it ranged from 0.08% ( total blood , gsm389907 ) to 4.36% of the total gene expression ( bronchial epithelium , gsm125353 ) . of 492 human sage catalogues tested , the cumulative expression of the imprinted genes constituted > 2% of the total gene expression in 21 and < 0.2% in 7 catalogues . the sage libraries with 10% most and 10% least cumulative and average expression of established and candidate imprinted gene subsets are listed in table 3 . in some samples , a major fraction of the cumulative expression of the imprinted genes for example , in the gsm125353 sage catalogue already mentioned above , 91.9% of the cumulative ( total ) gene expression of the assayed imprinted genes is represented by the single gene , namely , ptpn14 ( actttttcaa tag ) . similarly , in gsm383893 sage catalogue ( gallbladder tubular adenocarcinoma [ 17 , 23 ] ) , the same gene constitutes 86.6% of the cumulative ( total ) gene expression of the assayed imprinted genes . in many other sage catalogues , for example , in the gsm383840 sage catalogue ( mammary myoepithelium , cd10 cells ) , ptpn14 constitutes just 8.7% of the cumulative ( total ) gene expression of the assayed imprinted genes , equal to gnas gene ( attaacaaag tag ) . some imprinted genes were expressed almost ubiquitously through the samples : for example , genes ndufa4 , rpl22 , q8ne65 , ptpn14 , gnas , and rab1b ( supplementary table 3 ) . notably , in other cases , expression of the particular imprinted genes either was not detected at all in all 492 sage catalogues screened ( evx1 , acgcccgtgg tag ) , or was detected only occasionally ( supplementary table 3 ) . for example , gene dux2 ( aaggggtgga tag ) expression was detected only 3 times ( on a minimum level ) in 492 sage catalogues representing cell and tissue samples in a variety of physiological and pathological conditions : namely , in gsm383692 sage catalogue ( astrocytoma grade ii ) , gsm383867 sage catalogue ( colon carcinoma cell line [ 17 , 23 ] ) , and gsm383928 sage catalogue ( ovary preneoplasia cell line ) . similarly rare was the expression of fam75d1 ( detected only 3 times altogether ) , fam77d , ism1 , flj20464 , and q8nb05 ( detected only 5 times , in all cases on a minimum level ) . to assess variation in the expression of individual imprinted genes in the samples , the clustering analysis of the normalized expression profiles was performed using epclust ( expression profile data clustering and analysis ) software . for each dataset , the number of clusters was set to the lowest value yielding one cluster containing a solitary database entry ; 5 for cancer tissue , 6 for normal tissues and cells , 5 for cells cultured in vitro , and 2 for nontumorous disease tissue and cells ( figures 37 ) . notable diversity was observed in the transcription profiles represented by the individual clusters , with relatively high expression levels characteristic for just 1 - 2 or a higher number of the individual imprinted genes ( figures 4(a ) , 4(b)7(a ) , and 7(b ) ) . expectedly , in a few cases samples generated from the same tissues / cell types did fell into the same compact cluster of the distinct pattern ( figure 3 , figures 4(a ) , 4(b)7(a ) , and 7(b ) ) . however , in many other cases imprinted gene expression profiles of the same / similar tissue or cell types fall into different clusters . similarly , though in many cases imprinted gene expression profiles of the same / similar tissue or cell types fell into the closely matching area of the hierarchical tree built for the individual datasets ( clusters c , n , iv , and d ) ( figures 4(c ) , 5(c ) , 5(d ) , 6(c ) , and 7(c ) ) , in other cases notable variability was observed in the distribution of imprinted gene expression profiles of the same / similar tissue or cell types . for example , at k - mean clustering , small - size cluster 3 in cancer tissue dataset ( 3 entries ) is composed entirely of neuroblastoma samples ( figures 4(a ) and 4(b ) ) ; however , other entries representing tumors of the same histological properties   fell into cluster 1 ( composed of 141 entries in total ) . cluster 4 in the same dataset ( 12 entries ) is composed entirely of carcinoma samples , while cluster 2 ( 28 entries ) is composed of carcinoma samples predominantly ( 19 entries ) , with other samples representing astrocytoma ( 3 entries ) , glioblastoma multiforme ( 2 entries ) , cystadenoma ( 1 entry ) , rhabdosarcoma ( 1 entry ) , and unclassified breast cancer ( 2 entries ) . similarly , only one cluster in the normal tissue and cell dataset has a homogenous composition ( cluster 5 , 2 entries ) , matching both available sage libraries constructed from placenta ( gsm14849 , also designated gsm383945 ; gsm14750 , also designated gsm383947 ) ( figure 3 ) , with all other clusters composed of the samples of diverse origins . illustratively , this particular cluster brakes down ( i.e. , cluster content get redistributed to the clusters of the smaller size ) only if the number of k - mean clusters for the dataset is increased from the set value of 6 to 26 , while some other clusters break down more readily . in the hierarchical trees , most densely packed areas ( representing most similar transcription profiles ) are generally composed of the samples of the same / similar tissue or cell types . for example , one of the densest areas in four hierarchical trees built is composed of 19 samples matching bronchial brushings ( figures 5(c ) and 5(d ) ) , with all 5 other samples of the same origin falling into the nearest vicinity within the hierarchical tree ( figure 5(c ) ) . at the same time , some sage libraries representing the samples of the identical origin fell into the separate k - mean clusters and into well - separated areas of the hierarchical tree . this was observed , for example , for 3 available peripheral retina samples , from which gsm572 and gsm573   fell into cluster 3 , and gsm383968   fells into cluster 1 ( figure 4 ) . mechanism of genomic imprinting plays important , yet not fully understood role in many physiological processes : in particular , in the control of growth and development . since the identification of the first imprinted genes ( igf2 , igf2r , and h19 ) in mouse in 1991 , a large volume of information has been accumulated on the identity and biological function of imprinted genes both for homo sapiens and animal species ( mus musculus in particular ) . over the course of the decade , we witness an expansion of the list of the established imprinted genes [ 6 , 30 ] . it is most probable that novel candidate imprinted genes will be identified in the future , and features of the imprinted genes will be confirmed for some candidates . in the current study , a comprehensive list of the human imprinted genes and high - confidence gene candidates ( 203 entries total ) became a subject for a large - scale in silico gene expression profiling . available nucleotide sequences ( 174 genes and gene candidates ) have been utilized for the extraction of the appropriate short sage tags matching nlaiii anchoring enzyme , most common in generating sage libraries . notably , candidate imprinted gene q9nyi9 ( pparl ) did not bear nlaiii recognition sites . this limitation of the conventional sage protocol can generally be overcome by using an alternative anchoring enzyme . however , gene q9nyi9 does not bear recognition sites for anchoring enzymes sau3ai and rsai ( second and third most common in generating sage libraries ) as well , though it bears one for mmei utilized in longsage protocol . taken together , not 174 but 173 genes ( missing q9nyi9 ( pparl))including 53 established imprinted genes and 120 candidate imprinted genes the latter was matched the pool of 492 normalized sage catalogues representing libraries derived from human samples , constructed using nlaiii anchoring enzyme and together accounting for 35.97 million sage tags . collectively , these catalogues represent a comprehensive assay of tissues and cell types in physiological and a variety of pathological conditions . gene expression of imprinted genes was assessed in the normalized sage catalogues representing the transcriptomes of these samples , according to the straightforward algorithm of in silico analysis . as with nearly any other gene , expression of imprinted genes is not a constant , but rather a dynamic function of cell type and state . in the current study , a great variability was observed in both cumulative / total expression of the studied imprinted genes and that of the individual genes . the cumulative expression of 173 studied imprinted genes ranges from 0.08% ( total blood ) to 4.36% ( bronchial epithelium ) of the total gene expression ( table 3 ) . in some samples ( table 3 and supplementary table 2 ) , imprinted genes - associated proportion of the transcriptome is obviously above what is to be expected from such a limited group of genes , clearly reflecting the importance of the biological roles played by the latter . at the same time , overall expression of the imprinted genes was equal in the clusters of cancer tissues and normal tissue and cells ( clusters c and n , 0.95% for both clusters ) and lower for the cells cultured in vitro ( cluster iv , 0.77% ) . the current study apparently represents the first attempt to estimate an impact of imprinted genes on the total volume of the transcriptome . obvious biases affect an accuracy of the algorithm applied , suggesting both underestimation ( probable existence of yet unidentified imprinted genes , unavailable information on gene structure for some imprinted genes , absence of anchoring enzyme recognition sites for at least one gene ) and overestimation ( unconfirmed imprinting status of some of the candidate imprinted genes , sage tags matching more than one gene ; see table 1 ) of the relative size of the imprinted transcriptome . despite this , provided data on the estimated cumulative / total expression of the known imprinted genes ( their number well corresponding to the predicted number of imprinted genes in human genome [ 31 , 32 ] ) in a variety of tissues and cells is most interesting . until now , little information was available on the overall expression of imprinted genes in the cells of different types . it is generally believed that many imprinted genes are highly expressed in the developing and adult brain tissue , placenta , and undifferentiated stem cells . discrete studies identify certain highly expressed imprinted genes as the potential biomarkers of cancer subtypes [ 36 , 37 ] . in contrast , imprinted genes are known to be expressed on relatively low level in adult blood cells . this information is supported by the observed values of the cumulative expression of the imprinted genes through the screened samples ( table 3 and supplementary table 2 ) : cumulative expression of the imprinted genes is generally high in many assessed brain - derived samples and low in blood samples . it was also observed earlier that major upregulation of gene expression of the numerous imprinted genes is associated with early differentiation and development , rather than with undifferentiated status of stem cells [ 39 , 40 ] . concordantly , in the current study , all of the 13 sage libraries generated from undifferentiated embryonic stem cells ( escs)namely , lines hes3 , hes4 [ 17 , 23 , 41 ] , bg01 , h1 , h7 , h9 , h13 , h14 , hsf6 [ 17 , 23]uniformly demonstrate intermediate cumulative expression of the imprinted genes ( supplementary table 2 ) and fit closely in the hierarchical tree built for the corresponding cluster ( cluster iv ; figure 5(c ) ) . however , many samples with high cumulative expression of the imprinted genes do not fit into any of the groups listed above . important role of genomic imprinting in particular normal cell and cancer subtypes , suggested by high expression of these genes , thus should be a subject of the follow - up studies . expression of the candidate imprinted gene even - skipped homeobox 1 ( evx1 ) was not detected in any sample submitted to the analysis , while the expression of many more ( dux2 , fam75d1 , q8nb05 , flj20464 , ism1 , fam77d , and others ) was detected only in a few samples , always on a minimal level . in contrast , further imprinted genes ( ndufa4 , rpl22 , q8ne65 , gnas , ptpn14 , rab1b , and others ) were expressed in the majority of the samples screened , often on high level ( supplementary table 3 ) . illustratively , a notable variation in the cumulative expression of the imprinted genes and in the expression of individual imprinted genes is observed in the cells cultured in vitro , including cells of the same type ( e.g. , numerous medulloblastoma , glioblastoma multiforme , and breast carcinoma cell lines ) ( supplementary table 2 and figure 6 ) . this observation further supports earlier suggestion that cell culture conditions contribute to the maintenance or alteration of the imprinted gene expression [ 42 , 43 ] . taken together , a screening of the normalized expression profiles of a comprehensive panel of the established and candidate imprinted genes within the publicly available human sage datasets was performed in the current study : the first to estimate a prevalence of imprinted genes within the total human transcriptome in a large scale . this paper thus provides a useful reference on the relative size of the imprinted transcriptome and on the expression of the individual imprinted genes .
genomic imprinting is an epigenetic phenomenon that causes a differential expression of paternally and maternally inherited alleles of a subset of genes ( the so - called imprinted genes ) . 
 imprinted genes are distributed throughout the genome and it is predicted that about 1% of the human genes may be imprinted . 
 it is recognized that the allelic expression of imprinted genes varies between tissues and developmental stages . 
 the current study represents the first attempt to estimate a prevalence of imprinted genes within the total human transcriptome . in silico analysis of the normalized expression profiles of a comprehensive panel of 173 established and candidate human imprinted genes 
 was performed , in 492 publicly available sage libraries . 
 the latter represent human cell and tissue samples in a variety of physiological and pathological conditions . 
 variations in the prevalence of imprinted genes within the total transcriptomes ( ranging from 0.08% to 4.36% ) and expression profiles of the individual imprinted genes are assessed . 
 this paper thus provides a useful reference on the size of the imprinted transcriptome and expression of the individual imprinted genes .