# A Prevalence of Imprinted Genes within the Total Transcriptomes of Human Tissues and Cells

Authors: Sergey V. Anisimov
Journal: Molecular Biology International
 (2012)

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## Abstract

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.


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## Body

## 1. Introduction

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 [3–5]. Since then, the imprinting status was confirmed for numerous genes in Homo sapiensand Mus musculusgenomes, less for Bos taurus, Rattus norvegicus, Sus scrofa, Canis lupus familiaris, and Ovis aries; many more genes are considered candidates [6]. Functional significance of the genomic imprinting is not yet fully understood [7–9], while alterations in the expression of imprinted genes are linked to certain pathologies, including Angelman syndrome, Prader-Willi syndrome, and particular cancer subtypes. Genomic imprinting varies between species and tissues. Furthermore, it is a dynamic process and may vary depending on the developmental stage [10]. 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 [11]. 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 [12–15]. Numerous technical adaptations assured a development of similar techniques [16], 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 [17]. 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 [18]. It was therefore possible to estimate a prevalence of imprinted genes within the total human transcriptome.


## 2. Methods

### 2.1. Imprinted Gene Subsets

Established and candidate imprinted gene subset was assembled based on the Geneimprint resource (http://www.geneimprint.com/; credits to R.L. Jirtle) and Luedi et al. study [6]. Of the latter, 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 ([6], supplementary data) were utilized. Redundant entries have been excluded.


### 2.2. SAGE

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 [11]. 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. [19], were a subject to a “clean-up” procedure through which all clones containing ≤4 tags were excluded [20], with the remaining tags constituting the pool of “reliable tags.”


### 2.3. SAGE Tag Annotation

Established and candidate imprinted gene subset has matched CGAP (Cancer Genome Anatomy Project, NCI, NIH) SAGE Anatomic Viewer (SAV) applet [17]. 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).


### 2.4. Expression Profiling

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.


### 2.5. Clustering Analysis

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.


## 2.1. Imprinted Gene Subsets

Established and candidate imprinted gene subset was assembled based on the Geneimprint resource (http://www.geneimprint.com/; credits to R.L. Jirtle) and Luedi et al. study [6]. Of the latter, 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 ([6], supplementary data) were utilized. Redundant entries have been excluded.


## 2.2. SAGE

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 [11]. 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. [19], were a subject to a “clean-up” procedure through which all clones containing ≤4 tags were excluded [20], with the remaining tags constituting the pool of “reliable tags.”


## 2.3. SAGE Tag Annotation

Established and candidate imprinted gene subset has matched CGAP (Cancer Genome Anatomy Project, NCI, NIH) SAGE Anatomic Viewer (SAV) applet [17]. 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).


## 2.4. Expression Profiling

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.


## 2.5. Clustering Analysis

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.


## 3. Results

Established and candidate human imprinted gene subset (203 entries total) was assembled based on the Geneimprint resource and Luedi et al. study data [6]. Of the candidate imprinted genes identified in the latter, high-confidence gene candidates (predicted via Equbits Foresight and SMLR means [6]) 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 [18]. For a number of the candidate imprinted genes, a complete sequence was unavailable via GenBank or alternative databases (e.g., GenBank ID: 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 ID: 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.Table 1
SAGE tag annotation for established and candidate imprinted gene subset.
N
Gene symbol
Gene name
Aliases
Location
a
Status
Expressedallele
NlaIII tag
Notes
GenBank accession number
1
NDUFA4
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9 kDa
1p13.3
Candidate
Paternal
TTGGAGATCT
BC105295
2
GFI1
Growth-factor-independent 1 transcription repressor
ZNF163
1p22.1
Candidate
Paternal
TGTACCATAG
NM_001127215
3
NM019610
RNA-binding motif protein, X-linked-like 1 (RBMXL1), transcript variant 2
1p22.2
Candidate
Maternal
GCAGATTTAT
NM_019610
4
DIRAS3
DIRAS family, GTP-binding RAS-like 3
ARHI, NOEY2
1p31
Imprinted
Paternal
CAGAAAAAAA
*b
BC005362
5
BMP8B
Bone morphogenetic protein 8b
OP2, BMP8, MGC131757
1p35–p32
Candidate
Paternal
AGCAAAACTG
*
NM_001720
6
FUCA1
Fucosidase, alpha-L- 1, tissue
FUCA
1p36.11
Candidate
Paternal
CTATTTAGTT
NM_000147
7
TP73
TP73
P73
1p36.3
Imprinted
Maternal
TGGTACCGCC
NM_001126240
8
PRDM16
PR domain containing 16
MEL1
1p36.32
Candidate
Paternal
AGATTGATAT
NM_022114
9
PEX10
Peroxisomal biogenesis factor 10
1p36.32
Candidate
Maternal
GGAGGCGGCG
NM_002617
10
WDR8
WD repeat domain 8
1p36.32
Candidate
Maternal
TCGGTGCAGG
NM_017818
11
DVL1
Dishevelled, dsh homolog 1 (Drosophila)
DVL
1p36.33
Candidate
Maternal
GCCCGCAGGG
NM_004421
12
Q5EBL5
Family with sequence similarity 132, member A
FAM132A
1p36.33
Candidate
Maternal
GTTTCCAGGC
NM_001014980
13
TMEM52
Transmembrane protein 52
1p36.33
Candidate
Paternal
TTACACCGGC
NM_178545
14
HSPA6
Heat shock 70 kDa protein 6 (HSP70B′)
1q23.3
Candidate
Maternal
TATGAATTTT
NM_002155
15
PTPN14
Protein tyrosine phosphatase, nonreceptor type 14
PEZ
1q32.3
Candidate
Maternal
ACTTTTTCAA
*
BC017300
16
HIST3H2BB
Histone cluster 3, H2bb
1q42.13
Candidate
Maternal
AACTCCTTCG
*#c
NM_175055
17
OBSCN
Obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF
KIAA1556, KIAA1639
1q42.13
Candidate
Paternal
CTGAGCGCCG
*
NM_001098623
18
Q8NGX0
Olfactory receptor, family 11, subfamily L, member 1
OR11L1
1q44
Candidate
Paternal
AGAAGGAAAT
*
NM_001001959
19
VAX2
Ventral anterior homeobox 2
DRES93
2p13.3
Candidate
Maternal
GGCGATGGGG
NM_012476
20
OTX1
Orthodenticle homeobox 1
2p15
Candidate
Maternal
GCGGTTCCAG
BC007621
21
Q96PX6
Coiled-coil domain containing 85A
CCDC85A, KIAA1912
2p16.1
Candidate
Paternal
GCAGATATTC
R
d
NM_001080433
22
ABCG8
ATP-binding cassette, subfamily G (WHITE), member 8
2p21
Candidate
Maternal
GGCTCCAAAA
NM_022437
23
ZFP36L2
Zinc finger protein 36, C3H type-like 2
ERF2, TIS11D
2p21
Candidate
Maternal
TAGAAAGGCA
NM_006887
24
CYP1B1
Cytochrome P450, family 1, subfamily B, polypeptide 1
P4501B1
2p22.2
Candidate
Paternal
AATGCTTTTA
*
NM_000104
25
RPL22
Ribosomal protein L22
EAP
2q13
Candidate
Paternal
GATGCTGCCA
*
CR456873
26
TIGD1
Tigger transposable element derived 1
EEYORE
2q37.1
Candidate
Paternal
CGAAAAGCTT
R
BC063500
27
MYEOV2
Myeloma overexpressed 2
2q37.3
Candidate
Paternal
CAGACTTTTT
*
AF487338
28
FTHFD
10-Formyltetrahydrofolate dehydrogenase
ALDH1L1; DKFZp781N0997
3q21.3
Candidate
Maternal
TCTGCATCTT
BC027241
29
ZIC1
Zic family member 1 (odd-paired homolog,Drosophila)
ZIC, ZNF201
3q24
Candidate
Maternal
ATAATAGTGG
NM_003412
30
HES1
Hairy and enhancer of split 1, (Drosophila)
HHL, HRY, HES-1, bHLHb39, FLJ20408
3q29
Candidate
Paternal
CACTATATTT
NM_005524
31
FGFRL1
Fibroblast growth factor receptor-like 1
FHFR, FGFR5
4p16.3
Candidate
Maternal
AAAGTGCATC
NM_001004358
32
SPON2
Spondin 2, extracellular matrix protein
DIL1
4p16.3
Candidate
Paternal
TTATGGATCT
NM_001128325
33
Q9NYJ6
Immunoglobulin superfamily, member 9
IGSF9, 644ETD8, Dasm1, Kiaa1355-hp, NRT1, Ncaml, mKIAA1355
4q13.2
Candidate
Paternal
TTACTGGCCC
R
BC030141
34
NAP1L5
Nucleosome assembly protein 1-like 5
DRLM
4q22.1
Imprinted
Paternal
TAGCTTTTAG
NM_153757
35
DUX2
Double homeobox 2
4q35.2
Candidate
Paternal
AAGGGGTGGA
NM_012147
36
CDH18
Cadherin 18, type 2
CDH14, CDH24, CDH14L, EY-CADHERIN
5p14.3
Candidate
Paternal
ATCGAAACTG
NM_004934
37
ADAMTS16
ADAM metallopeptidase with thrombospondin type 1 motif, 16
FLJ16731, ADAMTS16s
5p15.32
Candidate
Maternal
TACCCCTGAA
*
AK122980
38
Q8TBP5
Family with sequence similarity 174, member A
FAM174A
5q21.1
Candidate
Paternal
ACCCAGCGGG
*
NM_198507
39
CSF2
Colony-stimulating factor 2 (granulocyte-macrophage)
GMCSF, MGC131935, MGC138897
5q23.3
Candidate
Maternal
GTGGGAGTGG
BC108724
40
BTNL2
Butyrophilin-like 2 (MHC class II associated)
SS2, BTLII, HSBLMHC1
6p21.32
Candidate
Maternal
GAAGGAAAGA
NM_019602
41
FAM50B
Family with sequence similarity 50, member B
X5L, D6S2654E
6p25.2
Imprinted
Paternal
CCTCAGTTTG
BC001261
42
C6orf117
Chromosome 6 open-reading frame 117
MRAP2
6q14.2
Candidate
Paternal
GCAAGCTGTT
NM_138409
43
HYMAI
Hydatidiform mole associated and imprinted (nonprotein coding)
NCRNA00020
6q24.2
Imprinted
Paternal
TATATATTGA
BC059359
44
PLAGL1
Pleiomorphic adenoma gene-like 1
ZAC, LOT1, ZAC1, MGC126275, MGC126276, DKFZp781P1017
6q24–q25
Imprinted
Paternal
ATCATAATGT
*
NM_001080951
45
SLC22A2
Solute carrier family 22 (organic cation transporter), member 2
OCT2, MGC32628
6q26
Imprinted
Maternal
AAAATTATAA
BC030978
46
SLC22A3
Solute carrier family 22 (extraneuronal monoamine transporter), member 3
EMT, EMTH, OCT3
6q26–q27
Imprinted
Maternal
TGCGCTAATC
AF078749
47
BRP44L
Brain protein 44-like
CGI-129, dJ68L15.3
6q27
Candidate
Paternal
CAGTGTATAT
BC000810
48
DDC
Dopa decarboxylase (aromatic L-amino acid decarboxylase)
AADC
7p12.2
Imprinted
Isoform Dependent
TGGCTAAATG
NM_000790
49
GRB10
Growth factor receptor-bound protein 10
RSS, IRBP, MEG1, GRB-IR, Grb-10, KIAA0207
7p12–p11.2
Imprinted
Isoform Dependent
TGCTTTGCTT
NM_001001549
50
GLI3
GLI family zinc finger 3
PHS, ACLS, GCPS, PAPA, PAPB, PAP-A, PAPA1, PPDIV
7p14.1
Candidate
Maternal
TAAATACATT
*
NM_000168
51
EVX1
Even-skipped homeobox 1
7p15.2
Candidate
Paternal
ACGCCCGTGG
NM_001989
52
HOXA5
Homeobox A5
HOX1C, HOX1.3, MGC9376
7p15.2
Candidate
Maternal
AGCCTGTTTA
BC013682
53
HOXA2
Homeobox A2
HOX1K
7p15.2
Candidate
Maternal
CATATTTTTT
*
NM_006735
54
HOXA3
Homeobox A3
HOX1E, MGC10155
7p15.2
Candidate
Maternal
CTCTTCCTCG
R
BC015180
55
HOXA11
Homeobox A11
HOX1I
7p15.2
Candidate
Maternal
GAGATAGCCC
BC040948
56
HOXA4
Homeobox A4
HOX1D
7p15.2
Candidate
Maternal
TGCTAAGAAT
NM_002141
57
TMEM60
Transmembrane protein 60
DC32, MGC74482, C7orf35
7q11.23
Candidate
Paternal
AATCTATCCT
NM_032936
58
PEG10
Paternally expressed 10
Edr, HB-1, Mar2, MEF3L, Mart2, RGAG3, KIAA1051
7q21
Imprinted
Paternal
GAAGTTATAA
NM_001040152
59
MAGI2
Membrane-associated guanylate kinase, WW and PDZ domain containing 2
AIP1, SSCAM, KIAA0705
7q21.11
Candidate
Maternal
TATTAATAGT
BC150277
60
PPP1R9A
Protein phosphatase 1, regulatory (inhibitor) subunit 9A
NRB1, NRBI, FLJ20068, KIAA1222, neurabin-I
7q21.3
Imprinted
Maternal
GAAGAGACAA
NM_017650
61
SGCE
Sarcoglycan, epsilon
ESG, DYT11
7q21–q22
Imprinted
Paternal
TTGGCAGTAT
*
NM_001099400
62
TFPI2
Tissue factor pathway inhibitor 2
PP5, REF1, TFPI-2, FLJ21164
7q22
Imprinted
Maternal
TGCTTTTAAC
NM_006528
63
MEST
Mesoderm-specific transcript homolog (mouse)
PEG1, MGC8703, MGC111102, DKFZp686L18234
7q32
Imprinted
Paternal
CTGAATGTAC
NM_002402
64
COPG2IT1
COPG2 imprinted transcript 1 (nonprotein coding)
CIT1, COPG2AS, FLJ41646, NCRNA00170, DKFZP761N09121
7q32
Imprinted
Paternal
GAGGGATGGC
*
AF038190
65
CPA4
Carboxypeptidase A4
CPA3
7q32
Imprinted
Maternal
TCTGTAAATC
*
BC052289
66
MESTIT1
MEST intronic transcript 1 (nonprotein coding)
PEG1-AS, NCRNA00040
7q32
Imprinted
Paternal
TGTAGTGGTG
NR_004382
67
KLF14
Kruppel-like factor 14
BTEB5
7q32.3
Imprinted
Maternal
TGGACTCTGG
NM_138693
68
SLC4A2
Solute carrier family 4, anion exchanger, member 2 (erythrocyte membrane protein band 3-like 1)
AE2, HKB3, BND3L, NBND3, EPB3L1
7q36.1
Candidate
Maternal
CCCCTCCCTC
*
NM_003040
69
FASTK
Fas-activated serine/threonine kinase
FAST
7q36.1
Candidate
Maternal
GGGGGTGGAT
NM_006712
70
PURG
Purine-rich element binding protein G
PURG-A, PURG-B, MGC119274
8p12
Candidate
Paternal
CTGAACAAAG
NM_001015508
71
DLGAP2
Discs, large (Drosophila) homolog-associated protein 2
DAP2, SAPAP2
8p23
Imprinted
Paternal
CCCCAGCCCC
*
NM_004745
72
Q8N9I4
FLJ37098 fis, clone BRACE2019004
8p23.1
Candidate
Paternal
CTAAGCGCAG
AK094417
73
FAM77D
Family with sequence similarity 77, member D
NKAIN3, FLJ39630
8q12.3
Candidate
Paternal
GTGCCCTACC
NM_173688
74
GPT
Glutamic-pyruvate transaminase (alanine aminotransferase)
GPT1, AAT1, ALT1
8q24.3
Candidate
Maternal
CCAAGTTCAC
NM_005309
75
KCNK9
Potassium channel, subfamily K, member 9
KT3.2, TASK3, K2p9.1, TASK-3, MGC138268, MGC138270
8q24.3
Imprinted
Maternal
CCAGGCACTC
*
AK090707
76
LY6D
Lymphocyte antigen 6 complex, locus D
E48
8q24.3
Candidate
Paternal
GAGATAAATG
BC031330
77
APBA1
Amyloid beta (A4) precursor protein-binding, family A, member 1
X11, D9S411E, MINT1, LIN10
9q21.11
Candidate
Paternal
TGTCTCCTTC
NM_001163
78
NM182505
Chromosome 9 open-reading frame 85
C9orf85, MGC61599, RP11-346E17.2
9q21.12
Candidate
Paternal
TAAAAATAAA
NM_182505
79
FAM75D1
Family with sequence similarity 75, member D1
FLJ46321
9q21.32
Candidate
Maternal
CCCCACAGGA
NM_001001670
80
ABCA1
ATP-binding cassette, subfamily A (ABC1), member 1
TGD, ABC1, CERP, ABC-1, HDLDT1, FLJ14958, MGC164864, MGC165011
9q31.1
Imprinted
Unknown
ATGGGGAGAG
*
AK024328
81
LMX1B
LIM homeobox transcription factor 1, beta
NPS1, LMX1.2, MGC138325, MGC142051
9q33.3
Candidate
Maternal
GGAGCCCAGC
*
NM_002316
82
EGFL7
EGF-like-domain, multiple 7
ZNEU1, MGC111117, VE-STATIN, RP11-251M1.2
9q34.3
Candidate
Paternal
GCACAGGCCA
NM_016215
83
PHPT1
Phosphohistidine phosphatase 1
PHP14, CGI-202, HSPC141, bA216L13.10, DKFZp564M173, RP11-216L13.10
9q34.3
Candidate
Maternal
GCCTATGGTC
NM_014172
84
NM144654
Chromosome 9 open-reading frame 116, transcript variant 2
C9orf116, FLJ13945, MGC29761, RP11-426A6.4
9q34.3
Candidate
Paternal
GGAAAGATGC
NM_144654
85
GATA3
GATA binding protein 3
HDR
10p14
Candidate
Paternal
AAGGATGCCA
*
BC003070
86
Q9H6Z8
FLJ21625 fis, clone COL08015
10q23.31
Candidate
Paternal
GCAGCAGCCT
AK025278
87
LDB1
LIM domain binding 1
CLIM2, NLI
10q24.32
Candidate
Maternal
TCCTGACCAC
NM_001113407
88
INPP5F V2
Inositol polyphosphate-5-phosphatase F
SAC2, hSAC2, MSTP007, MSTPO47, FLJ13081, KIAA0966, MGC59773, MGC131851
10q26.11
Imprinted
Paternal
AGATTGAGGC
NR_003252
89
C10orf93
Chromosome 10 open-reading frame 93
bB137A17.3, RP13-137A17.3
10q26.3
Candidate
Maternal
AACAAAATTA
BC044661
90
NKX6-2
NK6 homeobox 2
NK, NKX6B
10q26.3
Candidate
Maternal
ACCGAGAGCC
*
NM_177400
91
PAOX
Polyamine oxidase (exo-N4-amino)
PAO, DKFZp434J245
10q26.3
Candidate
Maternal
GAGACTCTGT
NM_152911
92
C10orf91
Chromosome 10 open-reading frame 91
bA432J24.4, RP11-432J24.4
10q26.3
Candidate
Maternal
GGTTCTCAGC
BC030794
93
VENTX2
VENT-like homeobox-2
NA88A, HPX42B, VENTX2
10q26.3
Candidate
Maternal
TGCTTTTAAA
AF068006
94
WT1-Alt trans
Wilms tumor 1
WT1, GUD, WAGR, WT33, WIT-2
11p13
Imprinted
Paternal
CTGGTATATG
BC032861
95
KCNQ1OT1
KCNQ1 overlapping transcript 1 (nonprotein coding)
LIT1, KvDMR1, KCNQ10T1, KvLQT1-AS, long QT intronic transcript 1
11p15
Imprinted
Paternal
AAATATTTAC
AF086011
96
KCNQ1DN
KCNQ1 downstream neighbor
BWRT, HSA404617
11p15.4
Imprinted
Maternal
GGACCCCAAA
AB039920
97
OSBPL5
Oxysterol binding protein-like 5
ORP5, OBPH1, FLJ42929
11p15.4
Imprinted
Maternal
GGGGATGGAT
NM_001144063
98
PKP3
Plakophilin 3
11p15.5
Candidate
Maternal
AACAGTCAAA
NM_007183
99
Q8N9U2
FLJ36520 fis, clone TRACH2002100
11p15.5
Candidate
Maternal
ACAAGTATTC
AK093839
100
IFITM1
Interferon-induced transmembrane protein 1 (9–27)
IFI17, LEU13, CD225
11p15.5
Candidate
Maternal
ACCATTGGAT
NM_003641
101
PHLDA2
Pleckstrin homology-like domain, family A, member 2
IPL, BRW1C, BWR1C, HLDA2, TSSC3
11p15.5
Imprinted
Maternal
AGCCCGCCGC
NM_003311
102
CDKN1C
Cyclin-dependent kinase inhibitor 1C (p57, Kip2)
BWS, WBS, p57, BWCR, KIP2
11p15.5
Imprinted
Maternal
CCCATCTAGC
NM_000076
103
SLC22A18
Solute carrier family 22, member 18
HET, ITM, BWR1A, IMPT1, TSSC5, ORCTL2, BWSCR1A, SLC22A1L, p45-BWR1A, DKFZp667A184
11p15.5
Imprinted
Maternal
CTGGGCCTCT
*
NM_002555
104
IGF2/INS
Insulin/insulin-like growth factor 2 (somatomedin A)
INSIGF, pp9974, C11orf43, FLJ22066, FLJ44734/ILPR, IRDN
11p15.5
Imprinted
Paternal
CTTGGGTTTT
BC011786
105
IGF2AS
Insulin-like growth factor 2 antisense
PEG8, MGC168198
11p15.5
Imprinted
Paternal
GAGGGCCGTT
AB030733
106
H19
H19, imprinted maternally expressed transcript (nonprotein coding)
ASM, BWS, ASM1, MGC4485, PRO2605, D11S813E
11p15.5
Imprinted
Maternal
GCCACCCCCT
*
BC007513
107
KCNQ1
Potassium voltage-gated channel, KQT-like subfamily, member 1
LQT, RWS, WRS, LQT1, SQT2, ATFB1, ATFB3, JLNS1, KCNA8, KCNA9, Kv1.9, Kv7.1, KVLQT1, FLJ26167
11p15.5
Imprinted
Maternal
GGCAGGAGAC
BC017074
108
B4GALNT4
Beta-1,4-N-acetyl-galactosaminyl transferase 4
FLJ25045
11p15.5
Candidate
Maternal
TGGAGCGTCC
NM_178537
109
RAB1B
RAB1B, member RAS oncogene family
11q13.2
Candidate
Maternal
TCAGGCATTT
BC071169
110
KBTBD3
Kelch repeat and BTB (POZ) domain containing 3
BKLHD3, FLJ30685
11q22.3
Candidate
Paternal
AAACTACAAA
AK092993
111
NTRI
Neurotrimin
NTM, HNT, IGLON2, MGC60329
11q25
Candidate
Paternal
TCCCTCTTCA
R
NM_016522
112
ABCC9
ATP-binding cassette, subfamily C (CFTR/MRP), member 9
SUR2, ABC37, CMD1O, FLJ36852
12p12.1
Candidate
Maternal
TGTCTTTAAA
*
BX537513
113
RBP5
Retinol binding protein 5, cellular
CRBP3, CRBPIII, CRBP-III
12p13.31
Imprinted
Maternal
CTTCCTGTTA
*
AK096947
114
HOXC4
Homeobox C4
HOX3E, CP19
12q13.13
Candidate
Maternal
GTACCTGCTG
NM_153633
115
HOXC9
Homeobox C9
HOX3B
12q13.13
Candidate
Maternal
TACGGCTCGC
BC032769
116
SLC26A10
Solute carrier family 26, member 10
12q13.3
Candidate
Maternal
ACCCTTGAAC
NM_133489
117
CDK4
Cyclin-dependent kinase 4
PSK-J3, CMM3
12q14.1
Candidate
Maternal
GAAGGAAGAA
*
BC005864
118
Q96AV8
E2F transcription factor 7
E2F7, FLJ12981
12q21.2
Candidate
Maternal
TAAACTGATT
BC016658
119
Q9HCM7
Fibrosin-1-like protein
FBRSL1, AUTS2L, KIAA1545, XTP9
12q24.33
Candidate
Maternal
TCAATCAGTG
NM_001142641
120
Q8N7V5
Proline-rich 20A
PRR20A, FLJ40296
13q21.1
Candidate
Maternal
ACTCACTGGA
*
NM_198441
121
FAM70B
Family with sequence similarity 70, member B
13q34
Candidate
Maternal
GTGCCTCTGT
NM_182614
122
FOXG1C
Forkhead box G1
HFK3
14q12
Candidate
Paternal
GAACTATATG
BC050072
123
PLEKHC1
Fermitin family (Drosophila) homolog 2
FERMT2, MIG2, UNC112, KIND2
14q22.1
Candidate
Paternal
GTTCAAAGAC
NM_001134999
124
DLK1
Delta-like 1 homolog (Drosophila)
DLK, FA1, ZOG, pG2, PREF1, Pref-1
14q32
Imprinted
Paternal
ATACAGAATA
*
BC013197
125
MEG3
Maternally expressed 3 (nonprotein coding)
GTL2, FP504, prebp1, PRO0518, PRO2160, FLJ31163, FLJ42589
14q32
Imprinted
Maternal
TGGGAAGTGG
AB032607
126
RTL1
Retrotransposon-like 1
PEG11
14q32.31
Candidate
Maternal
ACGGCCTGCA
NM_001134888
127
ATP10A
ATPase, class V, type 10A
ATPVA, ATPVC, ATP10C, KIAA0566
15q11.2
Imprinted
Maternal
GCCCCCAGAG
BC052251
128
PWCR1
Prader-Willi syndrome chromosome region 1
PET1, noncoding RNA in the Prader-Willi critical region
15q11.2
Imprinted
Paternal
TTGGTGAGGG
AF241255
129
NDN
Necdin homolog (mouse)
HsT16328
15q11.2–q12
Imprinted
Paternal
ACCTTGCTGG
BC008750
130
SNURF/SNRPN
SNRPN upstream reading frame/small nuclear ribonucleoprotein polypeptide N
SMN, PWCR, SM-D, RT-LI, HCERN3, SNRNP-N, FLJ33569, FLJ36996, FLJ39265, MGC29886, SNURF-SNRPN, DKFZp762N022, DKFZp686C0927, DKFZp761I1912, DKFZp686M12165
15q11.2–q12
Imprinted
Paternal
CCGCCTCCGG
BC000611
131
MAGEL2
MAGE-like 2
nM15, NDNL1
15q11–q12
Imprinted
Paternal
TAGCATTGTA
BC035839
132
MKRN3
Makorin ring finger protein 3
D15S9, RNF63, ZFP127, ZNF127, MGC88288
15q11–q13
Imprinted
Paternal
AAATAATTTA
NM_005664
133
UBE3A
Ubiquitin protein ligase E3A
AS, ANCR, E6-AP, HPVE6A, EPVE6AP, FLJ26981
15q11–q13
Imprinted
Maternal
CTGTAAAACA
BC002582
134
Q9P168
PRO2369
15q13.1
Candidate
Paternal
AGAACTCCAC
AF119879
135
SOX8
SRY (sex-determining region Y)-box 8
16p13.3
Candidate
Paternal
CAGCGTCTCC
BC031797
136
SALL1
Sal-like 1 (Drosophila)
HSAL1
16q12.1
Candidate
Maternal
ACATTTCTAG
R
BC113881
137
C16orf57
Chromosome 16 open-reading frame 57
16q13
Candidate
Maternal
GGATTTTAAT
BC004415
138
ACD
Adrenocortical dysplasia homolog (mouse)
PTOP, PIP1, TINT1, TPP1
16q22.1
Candidate
Maternal
CGGCAAAAAA
BC016904
139
FOXF1
Forkhead box F1
FKHL5, FREAC1, ACDMPV
16q24.1
Candidate
Maternal
TTCCTCCTCT
*
BC089442
140
ANKRD11
Ankyrin repeat domain 11
T13, LZ16, ANCO-1
16q24.3
Imprinted
Maternal
AAAGCTGACA
BC058001
141
Q8N206
FLJ36443 fis, clone THYMU2012891
FLJ36443 fis
16q24.3
Candidate
Maternal
ACATTCAGAA
AK093762
142
TMEM88
Transmembrane protein 88
FLJ20025
17p13.1
Candidate
Maternal
CTGGGCTTCG
NM_203411
143
PYY2
Peptide YY, 2 (seminal plasmin)
17q11.2
Candidate
Paternal
TTCACTCCCG
AF222904
144
HOXB3
Homeobox B3
HOX2G
17q21.32
Candidate
Maternal
AACTCAGCTC
NM_002146
145
HOXB2
Homeobox B2
HOX2H
17q21.32
Candidate
Maternal
AAGCACAAGC
NM_002145
146
Q8N8L1
FLJ39287 fis, clone OCBBF2011897
LOC100131170
17q25.3
Candidate
Paternal
GGGTCTGAGG
AK096606
147
FAM59A
Family with sequence similarity 59, member A
GAREM, Gm944, C18orf11
18q12.1
Candidate
Paternal
TGCAGAGAAA
NM_022751
148
BRUNOL4
Bruno-like 4
CELF4
18q12.2
Candidate
Maternal
GCTGTTCTTG
NM_001025087
149
TCEB3C
Transcription elongation factor B polypeptide 3C (elongin A3)
HsT829, TCEB3L2, elongin A3
18q21.1
Imprinted
Maternal
ACCTCCCAGG
*
NM_145653
150
Q8NE65
Zinc finger protein 738
ZNF738
19p13.11
Candidate
Paternal
TTGGTCAGGC
R
BC034499
151
Q8NB05
FLJ34424 fis, clone HHDPC2008279
19p13.2
Candidate
Paternal
TGCTCGGGAA
AK091743
152
PPAP2C
Phosphatidic acid phosphatase type 2C
PAP2C, LPP2
19p13.3
Candidate
Maternal
GTGTTCTTGG
NM_003712
153
TSH3
Teashirt zinc finger homeobox 3
TSHZ3, ZNF537, FLJ54422, KIAA1474
19q12
Candidate
Paternal
TTCTTATTTT
*
AK291466
154
CHST8
Carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8
GalNAc4ST1, GalNAc4ST
19q13.11
Candidate
Maternal
GTTTCCAGAG
*
NM_001127895
155
ZNF225
Zinc finger protein 225
MGC119735
19q13.31
Candidate
Paternal
TGGTATGTAT
NM_013362
156
ZNF229
Zinc finger protein 229
FLJ34222
19q13.31
Candidate
Maternal
TTGTAACCTC
NM_014518
157
ZNF264
Zinc finger protein 264
ZFP264
19q13.4
Imprinted
Maternal
GCTTCAGTGG
NM_003417
158
ZIM2/PEG3
ZIM2 zinc finger, imprinted 2/Paternally expressed 3
ZNF656/PW1, ZSCAN24, KIAA0287, DKFZp781A095
19q13.4
Imprinted
Paternal
TTTTCACCAT
BC037330
159
LILRB4
Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 4
LIR5, ILT3, HM18, CD85K
19q13.42
Candidate
Maternal
GGAAAATGGG
*
NM_001081438
160
ZNF550
Zinc finger protein 550
19q13.43
Candidate
Maternal
AGAAATGTAC
*
AK122867
161
CHMP2A
Chromatin-modifying protein 2A
VPS2A, VPS2, BC2
19q13.43
Candidate
Maternal
GGTGATGAGG
*
NM_014453
162
ZNF42
Zinc finger protein 42
MZF1, MZF1B, ZFP98, ZSCAN6
19q13.43
Candidate
Maternal
GTCAGAACAC
*
NM_003422
163
ISM1
Isthmin 1 homolog (zebrafish)
C20orf82
20p12.1
Candidate
Paternal
AATATTATCA
NM_080826
164
NNAT
Neuronatin
PEG5, MGC1439
20q11.2–q12
Imprinted
Paternal
CAGTTGTGGT
NM_005386
165
BLCAP
Bladder cancer-associated protein
BC10
20q11.2–q12
Imprinted
Isoform Dependent
CCTGTCCTTT
NM_006698
166
L3MBTL
L(3)mbt-like (Drosophila)
L3MBTL1, FLJ41181, KIAA0681, H-L(3)MBT, dJ138B7.3, DKFZp586P1522
20q13.12
Imprinted
Paternal
TGTGTATGTG
*
AB014581
167
GNAS
GNAS complex locus
AHO, GSA, GSP, POH, GPSA, NESP, GNAS1, PHP1A, PHP1B, C20orf45, MGC33735, dJ309F20.1.1, dJ806M20.3.3
20q13.3
Imprinted
Isoform Dependent
ATTAACAAAG
NM_000516
168
GNASAS
GNAS antisense RNA 1 (nonprotein coding)
SANG, NESPAS, GNAS1AS, NCRNA00075
20q13.32
Imprinted
Paternal
TCCATTAGAA
AJ251759
169
COL9A3
Collagen, type IX, alpha 3
IDD, MED, EDM3, FLJ90759, DJ885L7.4.1
20q13.33
Candidate
Maternal
AAGGAGCGGG
*
BC011705
170
C20orf20
Chromosome 20 open-reading frame 20
Eaf7, MRGBP, URCC4, MRG15BP, FLJ10914
20q13.33
Candidate
Maternal
ACCTCACTCT
BC009889
171
SIM2
Single-minded homolog 2 (Drosophila)
SIM, bHLHe15, MGC119447
21q22.13
Candidate
Paternal
AAGGAAGATT
*
NM_005069
172
DGCR6
DiGeorge syndrome critical region gene 6
22q11.21
Candidate
Paternal
CAGAAGAGGC
*
NM_005675
173
FLJ20464
Hypothetical protein FLJ20464
22q12.2
Candidate
Paternal
CGTGAAATTC
CR456348
SAGE tags annotated forNlaIII anchoring enzyme.
aEntries are sorted according to the established gene location.
b*: tag maps to other gene(s) according to CGAP (Cancer Genome Anatomy Project, NCI, NIH) SAGE Anatomic Viewer.
c#: highly repetitive tag according to CGAP SAGE Anatomic Viewer.
dR: unreliable/internal tag suggested by CGAP SAGE Anatomic Viewer is replaced with reliable 3′ end tag.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; GSM1515 = GSM383958; GSM85612 = GSM125353, etc.), 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).Table 2
Summary of SAGE catalogs analyzed.
Clusters
Number of SAGEcatalogsa
Number of SAGEtagsb
C (cancer tissue)
185
13,165,432
N (normal tissue and cells)
166
12,953,131
IV (cells culturedin vitro)
112
8,009,673
D  (nontumorous disease tissue and cells)
29
1,840,291
Total
492
35,968,527
All SAGE catalogs screened belong to GPL4 Gene Expression Omnibus database (GEO, NCBI) platform (Homo sapiens; NlaIII-anchoring enzyme).
aSAGE catalogs selected for analysis (see Supplementary Table 1 available online at doi:10.1155/2012/793506).
bNumber of tags subjected for analysis (with (A)10 tags excluded).Figure1 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 [21]) to 4.36% of the total gene expression (bronchial epithelium, GSM125353 [22]). 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.Table 3
The SAGE libraries with 10% most and 10% least cumulative and average expression of established and candidate imprinted genes subsets.
IDa
Primary IDb
SAGE library
Sample
Clusterc
Sumd
Averagee
Maxf
Top 10% libraries
76
301
GSM125353
Bronchial brushings, former smoker
N
43,563.92
251.81
40,054.67
4
6
GSM574
Central retina (macula)
N
33,159.24
191.67
26,692.01
142
427
GSM383793
Mammary gland, ductal carcinomain situ
C
29,781.50
172.15
25,275.20
29
104
GSM1730
Breast, ductal carcinomain situ
C
29,575.98
170.96
25,125.63
75
300
GSM125352
Bronchial brushings, former smoker
N
29,184.64
168.70
24,955.09
145
430
GSM383797
Mammary gland, ductal carcinoma
C
27,222.30
157.35
22,422.27
99
346
GSM194651
Oral biopsy
N
27,066.16
156.45
21,312.29
90
273
GSM112808
Neuroblastoma, primary tumor, stage 4S
C
25,944.14
149.97
17,365.83
55
155
GSM14753
Breast carcinoma metastasis to lung
C
23,941.22
138.39
20,247.08
143
428
GSM383794
Mammary gland, ductal carcinomain situ
C
23,570.83
136.25
18,977.90
30
105
GSM1731
Breast, ductal carcinomain situ
C
23,346.84
134.95
18,746.14
167
507
GSM383893
Gallbladder tubular adenocarcinoma
C
23,262.07
134.46
20,154.48
27
561
GSM384016
Vascular endothelium, hemangioma, benign hyperplasia
D
22,688.19
131.15
12,514.88
91
274
GSM112809
Neuroblastoma, primary tumor, stage 4S
C
22,633.99
130.83
11,595.94
28
101
GSM1516
Hemangioma tumor
C
22,622.41
130.77
12,500.82
100
347
GSM194652
Oral biopsy
N
21,279.52
123.00
11,932.33
146
440
GSM383807
Mammary gland, ductal carcinomain situ
C
20,570.15
118.90
16,071.37
62
433
GSM383800
Breast carcinoma cell line
IV
20,467.40
118.31
12,710.80
140
425
GSM383790
Mammary gland, ductal carcinoma
C
20,417.00
118.02
15,536.62
144
537
GSM383946
Whole body, fetal
N
20,274.33
117.19
9,698.99
149
443
GSM383812
Mammary gland, ductal carcinoma
C
20,036.49
115.82
13,377.93
5
21
GSM688
Breast, ductal carcinomain situ
C
19,968.16
115.42
15,538.48
118
416
GSM383775
Cortex, pooled sample
N
19,926.52
115.18
13,701.49
54
263
GSM85616
Bronchial epithelium
N
19,665.60
113.67
15,918.74
53
262
GSM85611
Bronchial epithelium
N
19,653.98
113.61
15,655.95
17
340
GSM194386
Metaplastic bronchial epithelium
D
19,376.31
112.00
14,577.13
81
306
GSM125358
Bronchial brushings, never smoker
N
19,369.05
111.96
14,172.30
137
509
GSM383895
Gallbladder
N
19,320.00
111.68
15,346.14
66
437
GSM383804
Breast carcinoma cell line
IV
19,247.28
111.26
9,389.55
31
106
GSM1733
Mammary gland, ductal invasivein situ carcinoma
C
19,126.03
110.56
14,594.68
92
276
GSM112812
Neuroblastoma, primary tumor, stage 4
C
18,914.78
109.33
12,349.04
3
5
GSM573
Peripheral retina
N
18,817.72
108.77
12,293.99
2
4
GSM572
Peripheral retina
N
18,781.47
108.56
8,727.76
96
331
GSM194377
Nonsmall cell lung cancer: squamous cell carcinomain situ
C
18,122.08
104.75
14,601.68
71
181
GSM14781
Brain desmoplastic medulloblastoma
C
18,106.48
104.66
11,244.70
68
439
GSM383806
Breast carcinoma cell line
IV
17,991.89
104.00
8,886.81
66
291
GSM125343
Bronchial brushings, former smoker
N
17,662.13
102.09
12,756.35
60
285
GSM125337
Bronchial brushings, current smoker
N
17,598.56
101.73
12,699.28
121
387
GSM383710
Ependymoma
C
17,524.27
101.30
10,222.49
51
260
GSM82458
Hippocampus
N
17,289.52
99.94
8,268.90
72
297
GSM125349
Bronchial brushings, former smoker
N
17,225.16
99.57
13,193.26
109
361
GSM296391
Lung biopsy
N
17,223.67
99.56
13,839.55
62
287
GSM125339
Bronchial brushings, current smoker
N
16,871.13
97.52
10,737.09
98
333
GSM194379
Nonsmall cell lung cancer: squamous cell carcinomain situ
C
16,572.81
95.80
12,713.33
74
299
GSM125351
Bronchial brushings, former smoker
N
16,456.57
95.12
11,137.34
70
295
GSM125347
Bronchial brushings, former smoker
N
16,306.85
94.26
11,835.62
7
216
GSM37212
Adrenal cortex affected by primary pigmented nodular adrenocortical disease
D
16,221.46
93.77
4,205.56
59
284
GSM125336
Bronchial brushings, current smoker
N
16,090.33
93.01
11,242.21
94
329
GSM194375
Nonsmall cell lung cancer: squamous cell carcinomain situ
C
15,715.04
90.84
11,679.51
Bottom 10% libraries
83
488
GSM383868
Colon carcinoma, cell line
IV
4,179.31
24.16
422.91
38
180
GSM14780
Gastric epithelial tissue from the antrum
N
4,169.95
24.10
1,615.39
84
204
GSM14807
Lung, poorly differentiated adenocarcinoma with lymphoplasmacytic infiltration
C
4,146.18
23.97
460.69
181
553
GSM383998
Gastroesophageal junction adenocarcinoma
C
4,141.08
23.94
1,351.60
1
7
GSM668
Kidney, embryonic cell line 293, uninduced cells
IV
4,119.01
23.81
920.45
4
120
GSM3244
AIDS-KS lesion
D
4,107.85
23.74
1,245.47
25
522
GSM383914
Lung, tumor associated (focal fibrosis and chronic inflammation)
D
4,103.62
23.72
606.45
30
121
GSM3245
CD4+ T cells
N
4,088.74
23.63
978.17
44
137
GSM14734
Medulloblastoma, cerebellum
C
4,081.98
23.60
804.90
56
162
GSM14760
Stomach, poorly differentiated carcinoma
C
4,081.16
23.59
1,340.95
54
153
GSM14751
Skin, melanoma
C
4,072.22
23.54
2,497.12
80
485
GSM383865
Colon carcinoma, cell line
IV
4,048.02
23.40
404.80
130
404
GSM383753
Medulloblastoma
C
3,987.16
23.05
1,069.73
82
487
GSM383867
Colon carcinoma, cell line
IV
3,961.32
22.90
506.24
12
71
GSM747
Colon, cancer cell line
IV
3,945.42
22.81
673.61
166
506
GSM383892
Gallbladder adenocarcinoma
C
3,909.95
22.60
651.66
115
372
GSM311354
CD15+ myeloid progenitor cells
N
3,859.27
22.31
1,108.11
52
261
GSM82459
Spermatozoa
N
3,814.90
22.05
1,288.15
50
259
GSM82243
Spermatozoa, pooled sample
N
3,811.16
22.03
1,429.18
94
328
GSM180670
Lymphocytes from children 1–4 years old (pooled samples)
N
3,794.27
21.93
843.17
81
486
GSM383866
Colon carcinoma, cell line
IV
3,746.12
21.65
378.40
39
183
GSM14784
Bone marrow
N
3,740.58
21.62
962.65
88
318
GSM136195
Cord blood-derived activated Th1 cells
N
3,668.81
21.21
777.29
20
423
GSM383787
Breast stroma, ductal carcinomain situ associated
D
3,663.78
21.18
334.67
38
234
GSM66698
HL-60 cells
IV
3,661.81
21.17
920.25
168
508
GSM383894
Gallbladder tubular adenocarcinoma
C
3,643.40
21.06
624.22
28
217
GSM37337
Primary bronchial epithelial cells
IV
3,604.30
20.83
804.84
177
546
GSM383970
Retinoblastoma
C
3,561.82
20.59
384.45
71
475
GSM383852
Cartilage chondrosarcoma cell line
IV
3,502.94
20.25
515.14
38
127
GSM7800
Primary gastric cancer, poorly differentiated (scirrhous type)
C
3,413.27
19.73
636.37
130
466
GSM383840
Mammary myoepithelium, CD10+ cells
N
3,364.85
19.45
291.33
39
235
GSM66712
HL-60 cells exposed to 2.45 GHz radiofrequency for 2 h
IV
3,293.77
19.04
982.36
97
344
GSM194649
Oral brushing
N
3,290.72
19.02
996.85
173
523
GSM383915
Lymph node, B-cell lymphoma
C
3,166.97
18.31
555.61
140
514
GSM383902
Leukocytes
N
2,991.69
17.29
439.34
65
436
GSM383803
Breast carcinoma cell line
IV
2,988.12
17.27
597.62
63
434
GSM383801
Breast carcinoma cell line
IV
2,977.71
17.21
297.77
21
84
GSM784
Gastric epithelial tissues
N
2,970.06
17.17
673.21
151
554
GSM384002
Stomach
N
2,813.83
16.26
654.38
170
515
GSM383903
Liver cholangiocarcinoma metastasis
C
2,736.32
15.82
1,575.46
29
578
GSM389908
Total blood after EPO treatment, pooled sample
D
2,684.90
15.52
747.69
68
175
GSM14775
Skin, primary malignant melanoma
C
2,612.99
15.10
653.25
40
236
GSM66714
HL-60 cells exposed to 2.45 GHz radiofrequency for 6 h
IV
1,879.82
10.87
639.52
143
533
GSM383937
Pancreas
N
1,813.11
10.48
278.94
83
308
GSM135389
Skeletal muscle, 5 days training young men
N
1,673.61
9.67
435.14
86
311
GSM135392
Skeletal muscle, detraining young men
N
1,670.43
9.66
510.41
165
576
GSM389906
Total blood, pooled sample
N
1,511.12
8.73
436.55
82
307
GSM135388
Skeletal muscle, pretraining young men
N
1,091.72
6.31
327.52
28
577
GSM389907
Total blood during EPO treatment, pooled sample
D
811.75
4.69
162.35
Indexes (GSM numbers) represent GEO database accession numbers for SAGE libraries (one accession number selected for redundant entries).
aID: listing within each cluster (see Supplementary Table 2).
bPrimary ID: listing within a full dataset (see Supplementary Table 1).
cClusters: C: cancer tissue; N: normal tissue and cells; IV: cells cultured in vitro; D: nontumorous disease tissue and cells.
dSum: cumulative (total) tag per million (tpm) value for SAGE tags matching established and candidate imprinted genes within the SAGE library.
eAverage: tpm value for SAGE tags matching established and candidate imprinted genes within the SAGE library.
fMax: maximum tpm value for SAGE tags matching established and candidate imprinted genes within the SAGE library.
Particular sum: average and maximum values could be recalculated to the fraction of the total gene expression by dividing tpm value to 1,000,000.
Entries are sorted according to the cumulative (total) tpm value.Figure 1
A schematic representation of the analyzed established (53, filled arrowheads) and candidate (120, empty arrowheads) imprinted genes distribution through the human genome. Chromosome layout is via NCBI (Build 37.2). Numbers next to some of the arrowheads indicate the number of entries per locus.Histogram of average ((a), (b), and (c)) and maximum ((d), (e), and (f)) tag per million (tpm) values of the pool of imprinted genes and gene candidates for the normalized SAGE catalogues: cancer tissue ((a), (d); 185 catalogues); normal tissues and cells ((b), (e); 166 catalogues); cells culturedin vitro ((c), (f); 112 catalogues). Corresponding histogram pairs are built following a sorting by the maximum value in the pool.
(a)
(b)
(c)
(d)
(e)
(f)In some samples, a major fraction of the cumulative expression of the imprinted genes was established by only a few highly abundant transcripts. 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, expression profile of the assayed imprinted genes was rather more balanced. For example, in the GSM383840 SAGE catalogue (mammary myoepithelium, CD10+ cells [24]), 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 [25]), GSM383867 SAGE catalogue (colon carcinoma cell line [17, 23]), and GSM383928 SAGE catalogue (ovary preneoplasia cell line [26]). 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 culturedin vitro, and 2 for nontumorous disease tissue and cells (Figures 3–7). 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 [27] 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 [17]) (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)) [22], 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 [28] fell into cluster 3, and GSM383968 [29] fells into cluster 1 (Figure 4).An example of gene expression pattern recognized by K-mean clustering analysis (normal tissue and cells, cluster 5). Graph line (a) and cluster contents (b). Vertical bars denote individual genes. Exponential shades of grey code (5 colors) are based on the normalized tpm values. GSM14749: first trimester placenta; GSM14750: full-term placenta. Imprinted genes with peak expression values in the cluster are indicated. PTPN14: protein tyrosine phosphatase, nonreceptor type 14; TFPI2: tissue factor pathway inhibitor 2; DLK1: delta-like 1 homolog (Drosophila).
(a)
(b)Gene expression patterns recognized by K-mean and hierarchical clustering analysis (cancer tissue, 185 SAGE catalogues). K-mean clustering analysis, (a) graph lines and (b) cluster contents; vertical bars denote individual genes. (c) Hierarchical cluster tree. Exponential shades of red code (15 colors) are based on the normalized tpm values.
(a)
(b)
(c)Gene expression patterns recognized by K-mean and hierarchical clustering analysis (normal tissue and cells, 166 SAGE catalogues). K-mean clustering analysis, (a) graph lines and (b) cluster contents; vertical bars denote individual genes. Arrowheads point out 3 SAGE libraries generated from peripheral retinal samples. (c) Hierarchical cluster tree, fragment (d) enlarged is highlighted. Exponential shades of red code (15 colors) are based on the normalized tpm values.
(a)
(b)
(c)
(d)Gene expression patterns recognized by K-mean and hierarchical clustering analysis (cells culturedin vitro, 112 SAGE catalogues). (a) K-mean clustering analysis, graph-lines and (b) cluster contents; vertical bars denote individual genes. (c) Hierarchical cluster tree. Arrowheads point out 13 SAGE libraries generated from undifferentiated embryonic stem cells (ESC). Exponential shades of red code (15 colors) are based on the normalized tpm values.
(a)
(b)
(c)Gene expression patterns recognized by K-mean and hierarchical clustering analysis (nontumorous disease tissue and cells, 29 SAGE catalogues). (a) K-mean clustering analysis, graph lines and (b) cluster contents; vertical bars denote individual genes. (c) Hierarchical cluster tree. Exponential shades of red code (15 colors) are based on the normalized tpm values.
(a)
(b)
(c)


## 4. Discussion

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 forHomo 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 [16]. However, gene Q9NYI9 does not bear recognition sites for anchoring enzymes Sau3AIandRsaI(second and third most common in generating SAGE libraries) as well, though it bears one for MmeIutilized in LongSAGE protocol. Taken together, not 174 but 173 genes (missing Q9NYI9 (PPARL))—including 53 established imprinted genes and 120 candidate imprinted genes—were annotated with the appropriate SAGE tags. The latter was matched  the pool of 492 normalized SAGE catalogues representing libraries derived from human samples, constructed using NlaIIIanchoring 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 (Table3). 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 Table1) 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 [33], placenta [34], and undifferentiated stem cells [35]. 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 [38]. 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 individual imprinted genes varies to even further extent in the samples screened. 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 culturedin 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.



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*Source: 793506-2012-09-11.xml*