Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2013 Article ID 105108 7 pageshttpdxdoiorg1011552013105108

Research ArticleThe Novelty of Human CancerTestis Antigen EncodingGenes in Evolution

Pavel Dobrynin12 Ekaterina Matyunina1 S V Malov2 and A P Kozlov12

1 The Biomedical Center Saint Petersburg 194044 Russia2 Dobzhansky Center for Genome Bioinformatics Saint Petersburg State University Saint Petersburg 190004 Russia

Correspondence should be addressed to Pavel Dobrynin pdobryningmailcom

Received 9 October 2012 Revised 16 January 2013 Accepted 13 February 2013

Academic Editor Ancha Baranova

Copyright copy 2013 Pavel Dobrynin et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In order to be inherited in progeny generations novel genes should originate in germ cells Here we suggest that the testes may playa special ldquocatalystrdquo role in the birth and evolution of new genes Cancertestis antigen encoding genes (CT genes) are predominantlyexpressed both in testes and in a variety of tumors By the criteria of evolutionary novelty the CT genes are indeed novel genesWe performed homology searches for sequences similar to human CT in various animals and established that most of the CT genesare either found in humans only or are relatively recent in their origin A majority of all human CT genes originated during or afterthe origin of Eutheria These results suggest relatively recent origin of human CT genes and align with the hypothesis of the specialrole of the testes in the evolution of the gene families

1 Introduction

In order to be inherited in progeny generations novel genesshould originate in germ cells Available data suggest thatthe generation of novel genes in germ cells is ongoingprocess for example the promiscuity of gene expressionin spermatogenic cells [1 2] Novel genes may originatethrough different mechanisms (retrogenes segmental dupli-cates chimeric and de novo emerged genes) but all of themare uniformly expressed in the testis ([3ndash8] reviewed in [9])These observations led us to suggest that testes may play aldquotissue catalystrdquo role in the birth and evolution of new genes[9] Previously we proposed the expression of evolutionarilynovel genes in tumors [10]

Cancertestis or cancergermline antigen genes are a classof genes with predominant expression in testis and in avariety of tumors with a significant exclusion of some CTantigens also expressed in the brain Here we set forth totest the hypothesis that cancertestis antigen genes shouldbe composed of evolutionarily new or young gene familyWe performed homology searches for sequences similar tohuman CT in various animals Additionally as an extensivetraffic of novel genes has been described for mammalian

X chromosome [3 6 11] we also performed this analysisseparately for genes located on this chromosome only

2 Methods

The list of CT antigens gene was retrieved from CT Database(httpwwwctalnccbr) and included 265 genes Amongthem there are 105 CT antigens that are encoded by theX chromosome (CT-X genes) and 105 that are located onvarious autosomes (autosome CT genes or non-XCT genes)Eight CT antigen encoding genes are located on the Ychromosome

To assess the evolutionary novelty of the studied groupof CT genes by searching orthologues for each of CT genesthe HomoloGenerelease 66 (httpwwwncbinlmnihgovhomologene) tool from NCBI website was used Homol-oGene is a database of both curated and computed geneorthologs and orthologues and now covers 21 organismsCurated orthologs include gene pairs from the MouseGenome Database (MGD) at the Jackson Laboratory theZebrafish Information (ZFIN) database at the University ofOregon and from published reports Computed orthologs

2 International Journal of Genomics

Table 1 Distribution of all human CT genes according to the origin of their orthologues in different taxa of human lineage

Taxa Chromosome names1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Eukaryote 1 1 1 2 1 1 1 1Opisthokonta 1 1Bilateria 1 1 1 2 2Coelomata 2 2 1 1Euteleostomi 4 4 2 1 1 2 2 3 1 2 1 1 1 1 3Amniota 3 2 2Eutheria 2 3 3 1 2 4 3 3 2 1 1 3 1 6 2 1 21 8Euarchontoglires 1 1 1 1 1 1 1 4Catarrhini 1 1 1 28Homininae 1 1 13Homo sapiens 1 33Total 11 7 5 4 2 7 1 10 4 6 4 4 1 2 5 3 7 4 8 5 3 2 105 8

and orthologues which are considered putative are identifiedfrom BLAST nucleotide sequence comparisons between allUniGene clusters for each pair of organisms [12] As an inputthe program uses gene name andor taxon name and theoutput is clusters of orthologues For this study the searchwas performed in several completely sequenced eukaryoticgenomes including H sapiens P troglodytes M mulatta Clupus B taurusMmusculus R norvegicus G gallus D rerioD melanogaster A gambiae C elegans S cerevisiae K lactisA gossypii S pombe M oryzae N crassa A thaliana Osativa and P falciparum

According to the origin of their orthologues in differenttaxa of human phylogeny the CT genes and all human geneswere distributed into 11 groupsThedifferences in distributionof CT genes and all human genes were assessed using thechi square test [13] Sheffersquos S method of multiple estimation([14 15] for counts see also [16]) was applied to define thedifference and to show stochastically that the origin of humanCT genes is substantially more recent than that for all humangenes

3 Results

The results obtained using HomoloGene tool applied tohuman CT genes are presented in Table 1 The full listof studied CT genes is present in Supplementary mate-rial (see Supplementary Material available online at httpdxdoiorg1011552013105108 HomoloGene assigned eachgene to a certain homology group which includes ortho-logues from different taxa within human lineage Of 265genes represented in CT Database 47 did not match anyhomology group probably because of the differences in thegene names making matches with HomoloGene databasedifficult HumanCT genes orthologues are widely distributedthroughout the human lineage For example for one CT-Xgene (FAM133A) the orthologues were found in all Eukary-ota and for two CT-X genes (MAGEC1 and SPANXN4)the orthologues were first found in Bilateria and for threeCT-X genes (ARX IL13RA and FAM46D) the time of

origin was placed in Euteleostomi There were substantiallylarger numbers of CT-X genes with orthologues emerging inEutheria Catarrhini and Homininae and of CT-X genes thatwere found exclusively in humans Interestingly there was aEutheria-specific subfamily TSPY1 composed of 8 CT genesand located on chromosome Y

Similarly searches for the orthologues were performed forall CT-X genes all autosomal CT genes all human CT genesand all annotated protein coding genes in human genome(assembly GRCh37) (Table 2 and Figure 1)

The results show that the proportion of autosomal CTgenes that has orthologues originated in Euteleostomi andin Eutheria (248 and 362 accordingly) is greater thanthat on chromosome X Only a few of autosomal CT genesare exclusive for humans We found that CT gene POTEB(prostate ovary testis-expressed protein on chromosome15 Ensembl ENSG00000233917) has a poorly characterizedhomologue (LOC100287399 Ensembl ENSG00000230031)that is according to HomoloGene criteria is exclusive toH sapiens This newly described homolog (LOC100287399Ensembl ENSG00000230031) has not been previously anno-tated as a gene of CT family

Among all annotated human protein coding genes theproportion of genes specific to humans only is very small(085) The list of these human-specific genes includes 163entries 33 of which are CT-X genes

For CT-X genes the distribution was different 314of CT-X genes (five CT45A genes twelve CT47A genesfifteen GAGE genes and four XAGE genes) are present inhumans only while 391 of CT-X genes have orthologuesthat emerged inCatarrhini orHomininaeThismeans that themajority (705) ofCT-X genes present in human genome areeither novel or relatively recent At the same time distributionof all genes located on X chromosome is similar to that for allhuman genes (see Supplementary Table IV)

Thedistribution of all humanCTgenes shows that 3073ofCTgenes have orthologues that originated inEutheriaThisproportion is larger than the proportion of all human geneswith pan-Eutherian orthologues (1641) Importantly 367of all human CT genes originated in Catarrhini Homininae

International Journal of Genomics 3

Table 2 CT-X genes autosomal CT genes all human CT genes and all annotated human genes with orthologues originated in different taxaof H sapiens lineage

Taxa CT-X genes Autosome CT genes All CT genes All human genesEukaryota 10 1 76 8 413 9 1519 2900Opisthokonta 19 2 092 2 321 613Bilateria 19 2 48 5 321 7 812 1549Coelomata 57 6 275 6 827 1579Euteleostomi 29 3 248 26 1330 29 3277 6256Amniota 67 7 321 7 839 1601Eutheria 200 21 362 38 3073 67 1641 3132Euarchontoglires 38 4 67 7 505 11 175 334Catarrhini 267 28 29 3 1422 31 266 507Homininae 124 13 19 2 688 15 238 454Homo sapiens 314 33 10 1 1560 34 085 163Total 1000 105 1000 105 10000 218 10000 19088

40302010

0

()

Human CT-X genes ()

40302010

0

()

Human autosomal CT genes ()

40302010

0

()

All human CT genes ()

Euka

ryot

a

Opi

sthok

onta

Bila

teria

Coelo

mat

a

Eute

leosto

mi

Amni

ota

Euth

eria

Euar

chon

togl

Cata

rrhi

ni

Hom

inin

ae

Hom

o

40302010

0

()

All annotated human genes ()

Figure 1 The proportions of CT-X genes autosomal CT genes all human CT genes and all annotated human genes with orthologuesoriginated in different taxa of H sapiens lineage

or humans Thus the majority of human CT genes (7248)originated during or after the emergence of Eutheria On theother side the majority of annotated human genes (7595)were older than Eutheria

A significance of the difference between distribution of allhuman genes and all humanCT genes according to the originof their orthologues in different taxa was confirmed bychisquare test (119875 value less than 10minus6)Moreover 95 confidence

region for the cumulative distribution function of CT humangenes displays that CT genes are stochastically younger ascompared to all human genes In other words the probabilitythat a gene randomly chosen from all human genes is youngerthan some fixed time 119879 is less than the probability that arandomly chosen CT gene is younger than119879Therefore thereis a significant bias in time of origin for human CT genes ascompared to all human genes If human CT genes would be

4 International Journal of Genomics

Euka

ryot

a

Opi

sthok

onta

Bila

teria

Coelo

mat

a

Eute

leosto

mi

Amni

ota

Euth

eria

Euar

chon

togli

res

Cata

rrhi

ni

Hom

o sa

pien

s

Hom

inin

ae

1

08

06

04

02

0

cdf for all human genesecdf for studied CT genes95 confidence band

Figure 2 Cumulative distribution function for all human genesand empirical distribution function for all CT human genes inaccordance with the origin of their orthologues in different taxawith 95 confidence bands cdfmdashcumulative distribution func-tion ecdfmdashempirical cumulative distribution function

obtained as a sample from some probabilistic distributionthe probability that CT human genes originated not earlierthan Catarrhini or Eutheria would be significantly higherthan the respective probability for census of all human genes(Figure 2) This statistical trial confirms that the origin ofhuman CT genes is relatively recent

4 Conclusion

Cancertestis antigen genes (CTA or CT genes) encode asubgroup of tumor antigens expressed predominantly intestis and various tumors CT antigens may be also expressedin placenta and in female germ cells [17ndash20] In additionsome CT antigens are expressed in the brain [21]

Experimentally human CT genes were discovered bya variety of immunological screening methods [22] sero-logical identification of antigens by recombinant expressioncloning (SEREX) [23] expression database analysis [2425] massively parallel signature sequencing [26] and otherapproaches The fact that many CT antigens have beenidentified using SEREX suggests that they are highly antigenic[23 27]

The first CT gene discovered was MAGEA1 that encodesfor an antigen of human melanoma [22] This gene belongsto a family of 12 closely related genes clustered at Xq28 Asecond cluster of MAGE genes MAGEB was discovered atXp213 and the third encoding MAGEC genes is located atXq26-27The expression ofMAGEA-MAGEC genes (MAGE-Isubfamily) is restricted to testis and cancer whereasmore dis-tantly related clusters MAGED-MAGEL (subfamily MAGE-II) are expressed in many normal tissuesMAGE-I genes areof relatively recent origin and MAGE-II genes are relativelymore ancient For example MAGE-D genes are conservedbetween man and mouse One of these genes corresponds

to the founder member of the family and the other MAGEgenes are retrogenes derived from the commonancestral gene[19 28 29]

To date CTD atabase (httpwwwctalnccbr) includes265CT genes More than half of them are located on X-chromosome (CT-X genes) [21] The analysis of the DNAsequence of the human X chromosome predicts that approx-imately 10 of the genes on the X chromosome are ofthe CT antigen type [30] Non-X CT genes are distributedthroughout the genome and are representedmainly by single-copy genes [19 27 31]

In normal testis CT-X genes are expressed in prolif-erating germ cells (spermatogonia) Non-X CT genes areexpressed during later stages of germ-cell differentiation thatis spermatocytes [19] Among human tumors CT antigensare expressed in melanoma bladder cancer lung cancerbreast cancer prostate cancer sarcoma ovarian cancer hepa-tocellular carcinoma hematologic malignancies and so forth[21 27 31 32] Genome-wide analysis of 153 cancertestisgenes expression has led to their classification into testis-restricted (119873 = 39) testisbrain-restricted (119873 = 14) andtestis-selective (119873 = 85) groups of genes the latter groupshowing some expression in nongermline tissuesThemajor-ity of testis-restricted genes belong to CT-X group (35 oftotal 39 testis-restricted groups) while non-X CT genes areexpressed in a less restrictive way [21]

Multiple CT antigens are often coexpressed in tumorssuggesting that this expression program is coordinated forentire family [19 23 33] CT gene expression is controlledby epigenetic mechanisms which include DNA methyla-tion and histone posttranslational modifications [31] Othermechanisms of CT gene regulation include sequence-specifictranscription factors and signal transduction pathways suchas activated tyrosine kinases [34]

The functions of CT-X genes are largely unknown Onthe contrary more is known about functions of non-X CTgenes which are associated with meiosis gametogenesis andfertilization Non-X CTs are also more conserved duringevolution [21 27 31 32]

CT-X genes tend to form recently expanded gene familiesmany with nearly identical gene copies [17ndash20 26 32 35]

The prevalence of large highly homologous invertedrepeats (IRs) containing testes genes on the X- and Y-chromosomes was described in humans and great apes [3637] CT-X gene families are also located in direct or invertedrepeats [20]

The study of clusters of homologous genes originated bygene duplication roughly after the divergence of the humanand rodent lineages discovered several families of CT genesamong recent duplicates [38]

In the other paper the authors also studied recentduplications in the human genome and found that CTgenes were represented in this gene set including the familyof PRAME (preferentially expressed antigen of melanoma)genes located on chromosome 1 and expressed in the testisand in a large number of tumors [39] Duplicated PRAMEgenes are hominid specific having arisen in human genomesince the divergence from chimps PRAME gene familyalso expanded in other Eutheria Chimp and mouse have

International Journal of Genomics 5

orthologous PRAME gene clusters on their chromosomes 1and 4 respectively [39 40]

Rapid evolution of cancertestis genes has been demon-strated on the X chromosome In particular the comparisonof human chimp orthologues of these genes has shown thatthey diverge faster and undergo stronger positive selectionthan those on the autosomes or than control genes on eitherX chromosome or autosomes [41]

SPANX-AD gene subfamily of cancertestis-specific anti-gens evolved in the common ancestor of the hominoidlineage after its separation from orangutan Southern blotand database analyses have detected SPANX sequences onlyin primates [17] The coding sequences of the SPANXgenes evolved rapidly faster than their introns and the51015840 untranslated regions with accelerated rates of substi-tutions in both synonymous and nonsynonymous codonpositions The mechanism of SPANX genes expansion wassegmental DNA duplications with evidence of positiveselection SPANX-N is the ancestral form from which theSPANX-AD subfamily evolved in the common ancestor ofhominoids approximately 7 MYA [35 42] SPANX genesare expressed in cancer cells and highly metastatic celllines from melanomas bladder carcinomas and myelomas[35]

The GAGE cancertestis antigen gene family containsat least 16 genes which are encoded by an equal numberof tandem repeats All GAGE genes are located at Xp1123GAGE genes are highly identical and evolved under positiveselection that supports their recent origin [43 44]

The XAGE family of cancertestis antigen genes belongsto superfamily of GAGE-like CT genes It is located on chro-mosomeXp1121-Xp1122ThreeXAGE genes are described aswell as several splice variants of XAGE-1 [45 46]

CT45 gene family was discovered by massively parallelsignature sequencing It includes six highly similar (gt98)genes that are cluctered in tandem on chromosome Xq263CT45 antigen is expressed in Hodgkinrsquos lymphoma and inother human tumors [26 47ndash49]

CT47 cancertestis gene family is located on chromosomeXq24 Among normal tissues it is expressed in the testis and(weakly) in placenta and brain In tumors its expression wasfound in lung cancer and esophageal cancerTheCT47 familymember is characterized by high (gt98) sequence homologyChimp is the only other species in which a gene homologousto CT47 was found by other authors [20]

Our work is the first systematic study of the evolutionarynovelty of the whole class of CT genes To assess the evolu-tionary novelty of CT genes we applied the HomoloGenetool of NCBI To construct the clusters of orthologuesthe HomoloGene program uses information from blastpphylogenetic analyses and syntheny information when it ispossible Cutoffs on bits per position and Ks values are setto prevent unlikely ldquoorthologsrdquo from being grouped togetherThese cutoffs are calculated based on the respective scoredistribution for the given groups of organisms [12]

We searched for orthologues of each of CT genes amongannotated genes in several completely sequenced eukaryoticgenomes and built distributions of all CT-X genes all autoso-mal CT genes all human CT genes and all annotated protein

coding genes from human genome according to the origin oftheir orthologues in 11 taxa of human lineage

We have shown that 314 of CT-X genes are exclusive forhumans and 391ofCT-Xgenes have orthologues originatedin Catarrhini or Homininae Thereby the majority of humanCT-X genes (705) are novel or recent in its origin Our dataare in good correspondence with evidence obtained by othergroups on rapid expansion of certain CT-X gene families andhigh homology of their members which suggest their recentorigin

Altogether 367 of all human CT genes originated inCatarrhini Homininae and humansWe have also found that3073 of all human CT genes originated in Eutheria TheseCT genes acquired functions in Eutheria This indicates theimportance of processes in which tumors and CT antigenswere involved during the evolution of Eutheria CT genesoriginated in Eutheria are located mostly on autosomes CTgenes originated in Catarrhini Homininae and humans arelocated predominantly on X chromosome This difference isprobably related to evolution of mammalian X chromosomesince the origin of Eutheria [50] especially to the acquisitionof its special role in the origin of novel genes [9]

Thus the majority of CT-X genes are either novel oryoung for humans and the majority of all human CT genes(7248) originated during or after the origin of EutheriaThese results suggest that the whole class of human CT genesis relatively evolutionarily new

In its turn this conclusion confirms our prediction aboutexpression of evolutionary recent and novel genes in tumors[10] The expression of cancertestis genes in tumors is thena natural phenomenon not aberrant process as suggested bymany authors (eg [19 27 32 34 40])

References

[1] E E Schmidt ldquoTranscriptional promiscuity in testesrdquo CurrentBiology vol 6 no 7 pp 768ndash769 1996

[2] K C Kleene ldquoSexual selection genetic conflict selfish genesand the atypical patterns of gene expression in spermatogeniccellsrdquo Developmental Biology vol 277 no 1 pp 16ndash26 2005

[3] E Betran K Thornton and M Long ldquoRetroposed new genesout of the X in DrosophilardquoGenome Research vol 12 no 12 pp1854ndash1859 2002

[4] C A Paulding M Ruvolo and D A Haber ldquoThe Tre2(USP6) oncogene is a hominoid-specific generdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 5 pp 2507ndash2511 2003

[5] X She Z Cheng S Zollner D M Church and E E EichlerldquoMouse segmental duplication and copy number variationrdquoNature Genetics vol 40 no 7 pp 909ndash914 2008

[6] M T Levine C D Jones A D Kern H A Lindfors andD J Begun ldquoNovel genes derived from noncoding DNA inDrosophila melanogaster are frequently X-linked and exhibittestis-biased expressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 103 no 26 pp9935ndash9939 2006

[7] T J A J Heinen F Staubach D Haming and D Tautz ldquoEmer-gence of a new gene from an intergenic regionrdquoCurrent Biologyvol 19 no 18 pp 1527ndash1531 2009

6 International Journal of Genomics

[8] H Kaessmann N Vinckenbosch and M Long ldquoRNA-based gene duplication mechanistic and evolutionary insightsrdquoNature Reviews Genetics vol 10 no 1 pp 19ndash31 2009

[9] H Kaessmann ldquoOrigins evolution and phenotypic impact ofnew genesrdquo Genome Research vol 20 no 10 pp 1313ndash13262010

[10] A P Kozlov ldquoThe possible evolutionary role of tumors in theorigin of new cell typesrdquo Medical Hypotheses vol 74 no 1 pp177ndash185 2010

[11] J J Emerson H Kaessmann E Betran and M Long ldquoExten-sive gene traffic on the mammalian X chromosomerdquo Sciencevol 303 no 5657 pp 537ndash540 2004

[12] EW Sayers T Barrett D A Benson et al ldquoDatabase resourcesof the National Center for Biotechnology Informationrdquo NucleicAcids Research vol 40 pp D13ndashD25 2012

[13] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg

[14] H SheffeThe Analysis of Variance Wiley New York NY USA1959

[15] H Sheffe ldquoMultiple testing versus multiple estimationImproper confidence sets Estimation of directions and ratiosrdquoThe Annals of Mathematical Statistics vol 41 no 1 pp 1ndash291970

[16] L A Goodman ldquoSimultaneous confidence intervals for con-trasts among multinomial populationsrdquo The Annals of Mathe-matical Statistics vol 35 pp 716ndash725 1964

[17] A J W Zendman J Zschocke A A Van Kraats et alldquoThe human SPANX multigene family genomic organizationalignment and expression in male germ cells and tumor celllinesrdquo Gene vol 309 no 2 pp 125ndash133 2003

[18] A J W Zendman D J Ruiter and G N P Van MuijenldquoCancertestis-associated genes identification expression pro-file and putative functionrdquo Journal of Cellular Physiology vol194 no 3 pp 272ndash288 2003

[19] A J G Simpson O L Caballero A Jungbluth Y T Chen andL J Old ldquoCancertestis antigens gametogenesis and cancerrdquoNature Reviews Cancer vol 5 no 8 pp 615ndash625 2005

[20] Y T Chen C Iseli C A Yenditti L J Old A J G Simpsonand C V Jongeneel ldquoIdentification of a new cancertestis genefamily CT47 among expressed multicopy genes on the humanX chromosomerdquoGenes Chromosomes and Cancer vol 45 no 4pp 392ndash400 2006

[21] O Hofmann O L Caballero B J Stevenson et al ldquoGenome-wide analysis of cancertestis gene expressionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 51 pp 20422ndash20427 2008

[22] P Van Der Bruggen C Traversari P Chomez et al ldquoA geneencoding an antigen recognized by cytolytic T lymphocytes ona human melanomardquo Science vol 254 no 5038 pp 1643ndash16471991

[23] U Sahin O Tereci H Schmitt et al ldquoHuman neoplasms elicitmultiple specific immune responses in the autologous hostrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11810ndash11813 1995

[24] M J ScanlanCMGordon BWilliamson et al ldquoIdentificationof cancertestis genes by database mining and mRNA expres-sion analysisrdquo International Journal of Cancer vol 98 no 4 pp485ndash492 2002

[25] M J Scanlan A J Simpson and L J Old ldquoThe cancertestisgenes review standardization and commentaryrdquo Cancer Im-munity vol 4 article 1 2004

[26] Y T Chen M J Scanlan C A Venditti et al ldquoIdentificationof cancertestis-antigen genes by massively parallel signaturesequencingrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 102 no 22 pp 7940ndash79452005

[27] Y H Cheng E W P Wong and C Y Cheng ldquoCancertestis(CT) antigens carcinogenesis and spermatogenesisrdquo Spermato-genesis vol 1 pp 209ndash220 2011

[28] P Chomez O De Backer M Bertrand E De Plaen T Boonand S Lucas ldquoAn overview of the MAGE gene family withthe identification of all human members of the familyrdquo CancerResearch vol 61 no 14 pp 5544ndash5551 2001

[29] Y Katsura and Y Satta ldquoEvolutionary history of the cancer Im-munity antigen MAGE gene familyrdquo PLoS ONE vol 6 no 6Article ID e20365 2011

[30] M T Ross D V Grafham A J Coffey et al ldquoThe DNAsequence of the human X chromosomerdquo Nature vol 434 pp325ndash337 2005

[31] E Fratta S Coral A Covre et al ldquoThe biology of cancertestis antigens putative function regulation and therapeuticpotentialrdquoMolecular Oncology vol 5 no 2 pp 164ndash182 2011

[32] O L Caballero and Y T Chen ldquoCancertestis (CT) antigenspotential targets for immunotherapyrdquo Cancer Science vol 100no 11 pp 2014ndash2021 2009

[33] U Sahin O Tureci Y T Chen et al ldquoExpression of multiplecancertestis antigens in breast cancer and melanoma basisfor polyvalent CT vaccine strategiesrdquo International Journal ofCancer vol 78 pp 387ndash389 1998

[34] S N Akers K Odunsi and A R Karpf ldquoRegulation of cancergermline antigen gene expression implications for cancerimmunotherapyrdquo Future Oncology vol 6 no 5 pp 717ndash7322010

[35] N Kouprina MMullokandov I B Rogozin et al ldquoThe SPANXgene family of cancertestis-specific antigens rapid evolutionand amplification in African great apes and hominidsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 9 pp 3077ndash3082 2004

[36] H Skaletsky T Kuroda-Kawaguchl P J Minx et al ldquoThemale-specific region of the human Y chromosome is a mosaic ofdiscrete sequence classesrdquo Nature vol 423 no 6942 pp 825ndash837 2003

[37] P E Warburton J Giordano F Cheung Y Gelfand and GBenson ldquoInverted repeat structure of the humangenome theX-chromosome contains a preponderance of large highly homol-ogous inverted repeated that contain testes genesrdquo GenomeResearch A vol 14 no 10 pp 1861ndash1869 2004

[38] IHGSC ldquoInternational human genome sequencing consor-tiumrdquo Nature vol 431 pp 931ndash945 2004

[39] Z Birtle L Goodstadt and C Ponting ldquoDuplication andpositive selection among hominin-specific PRAME genesrdquoBMC Genomics vol 6 article 120 2005

[40] T C Chang Y Yang H Yasue A K Bharti E F Retzel andWS Liu ldquoThe expansion of the PRAME gene family in EutheriardquoPLoS ONE vol 6 no 2 Article ID e16867 2011

[41] B J Stevenson C Iseli S Panji et al ldquoRapid evolution ofcancertestis genes on the X chromosomerdquo BMCGenomics vol8 article 129 2007

[42] N Kouprina V N Noskov A Pavlicek et al ldquoEvolutionarydiversification of SPANX-N sperm protein gene structure andexpressionrdquo PLoS ONE vol 2 no 4 article e359 2007

International Journal of Genomics 7

[43] Y Liu Q Zhu and N Zhu ldquoRecent duplication and positiveselection of the GAGE gene familyrdquo Genetica vol 133 no 1 pp31ndash35 2008

[44] M F Gjerstorff andH J Ditzel ldquoAn overview of the GAGE can-certestis antigen family with the inclusion of newly identifiedmembersrdquo Tissue Antigens vol 71 no 3 pp 187ndash192 2008

[45] A J W Zendman A A Van Kraats U H Weidle D J Ruiterand G N P Van Muijen ldquoThe XAGE family of cancertestis-associated genes alignment and expression profile in normaltissues melanoma lesions and Ewingrsquos sarcomardquo InternationalJournal of Cancer vol 99 no 3 pp 361ndash369 2002

[46] S Sato Y Noguchi N Ohara et al ldquoIdentification of XAGE-1 isoforms predominant expression of XAGE-1b in testis andtumorsrdquo Cancer Immunity vol 7 article 5 2007

[47] Y T Chen M Hsu P Lee et al ldquoCancertestis antigen CT45analysis of mRNA and protein expression in human cancerrdquoInternational Journal of Cancer vol 124 no 12 pp 2893ndash28982009

[48] Y T Chen A Chadburn P Lee et al ldquoExpression of cancertestis antigen CT45 in classical Hodgkin lymphoma and otherB-cell lymphomasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 7 pp 3093ndash3098 2010

[49] H J Heidebrecht A Claviez M L Kruse et al ldquoCharacteriza-tion and expression of CT45 in Hodgkinrsquos lymphomardquo ClinicalCancer Research vol 12 pp 4804ndash4811 2006

[50] B T Lahn and D C Page ldquoFour evolutionary strata on thehumanX chromosomerdquo Science vol 286 no 5441 pp 964ndash9671999

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Microbiology

2 International Journal of Genomics

Table 1 Distribution of all human CT genes according to the origin of their orthologues in different taxa of human lineage

Taxa Chromosome names1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Eukaryote 1 1 1 2 1 1 1 1Opisthokonta 1 1Bilateria 1 1 1 2 2Coelomata 2 2 1 1Euteleostomi 4 4 2 1 1 2 2 3 1 2 1 1 1 1 3Amniota 3 2 2Eutheria 2 3 3 1 2 4 3 3 2 1 1 3 1 6 2 1 21 8Euarchontoglires 1 1 1 1 1 1 1 4Catarrhini 1 1 1 28Homininae 1 1 13Homo sapiens 1 33Total 11 7 5 4 2 7 1 10 4 6 4 4 1 2 5 3 7 4 8 5 3 2 105 8

and orthologues which are considered putative are identifiedfrom BLAST nucleotide sequence comparisons between allUniGene clusters for each pair of organisms [12] As an inputthe program uses gene name andor taxon name and theoutput is clusters of orthologues For this study the searchwas performed in several completely sequenced eukaryoticgenomes including H sapiens P troglodytes M mulatta Clupus B taurusMmusculus R norvegicus G gallus D rerioD melanogaster A gambiae C elegans S cerevisiae K lactisA gossypii S pombe M oryzae N crassa A thaliana Osativa and P falciparum

According to the origin of their orthologues in differenttaxa of human phylogeny the CT genes and all human geneswere distributed into 11 groupsThedifferences in distributionof CT genes and all human genes were assessed using thechi square test [13] Sheffersquos S method of multiple estimation([14 15] for counts see also [16]) was applied to define thedifference and to show stochastically that the origin of humanCT genes is substantially more recent than that for all humangenes

3 Results

The results obtained using HomoloGene tool applied tohuman CT genes are presented in Table 1 The full listof studied CT genes is present in Supplementary mate-rial (see Supplementary Material available online at httpdxdoiorg1011552013105108 HomoloGene assigned eachgene to a certain homology group which includes ortho-logues from different taxa within human lineage Of 265genes represented in CT Database 47 did not match anyhomology group probably because of the differences in thegene names making matches with HomoloGene databasedifficult HumanCT genes orthologues are widely distributedthroughout the human lineage For example for one CT-Xgene (FAM133A) the orthologues were found in all Eukary-ota and for two CT-X genes (MAGEC1 and SPANXN4)the orthologues were first found in Bilateria and for threeCT-X genes (ARX IL13RA and FAM46D) the time of

origin was placed in Euteleostomi There were substantiallylarger numbers of CT-X genes with orthologues emerging inEutheria Catarrhini and Homininae and of CT-X genes thatwere found exclusively in humans Interestingly there was aEutheria-specific subfamily TSPY1 composed of 8 CT genesand located on chromosome Y

Similarly searches for the orthologues were performed forall CT-X genes all autosomal CT genes all human CT genesand all annotated protein coding genes in human genome(assembly GRCh37) (Table 2 and Figure 1)

The results show that the proportion of autosomal CTgenes that has orthologues originated in Euteleostomi andin Eutheria (248 and 362 accordingly) is greater thanthat on chromosome X Only a few of autosomal CT genesare exclusive for humans We found that CT gene POTEB(prostate ovary testis-expressed protein on chromosome15 Ensembl ENSG00000233917) has a poorly characterizedhomologue (LOC100287399 Ensembl ENSG00000230031)that is according to HomoloGene criteria is exclusive toH sapiens This newly described homolog (LOC100287399Ensembl ENSG00000230031) has not been previously anno-tated as a gene of CT family

Among all annotated human protein coding genes theproportion of genes specific to humans only is very small(085) The list of these human-specific genes includes 163entries 33 of which are CT-X genes

For CT-X genes the distribution was different 314of CT-X genes (five CT45A genes twelve CT47A genesfifteen GAGE genes and four XAGE genes) are present inhumans only while 391 of CT-X genes have orthologuesthat emerged inCatarrhini orHomininaeThismeans that themajority (705) ofCT-X genes present in human genome areeither novel or relatively recent At the same time distributionof all genes located on X chromosome is similar to that for allhuman genes (see Supplementary Table IV)

Thedistribution of all humanCTgenes shows that 3073ofCTgenes have orthologues that originated inEutheriaThisproportion is larger than the proportion of all human geneswith pan-Eutherian orthologues (1641) Importantly 367of all human CT genes originated in Catarrhini Homininae

International Journal of Genomics 3

Table 2 CT-X genes autosomal CT genes all human CT genes and all annotated human genes with orthologues originated in different taxaof H sapiens lineage

Taxa CT-X genes Autosome CT genes All CT genes All human genesEukaryota 10 1 76 8 413 9 1519 2900Opisthokonta 19 2 092 2 321 613Bilateria 19 2 48 5 321 7 812 1549Coelomata 57 6 275 6 827 1579Euteleostomi 29 3 248 26 1330 29 3277 6256Amniota 67 7 321 7 839 1601Eutheria 200 21 362 38 3073 67 1641 3132Euarchontoglires 38 4 67 7 505 11 175 334Catarrhini 267 28 29 3 1422 31 266 507Homininae 124 13 19 2 688 15 238 454Homo sapiens 314 33 10 1 1560 34 085 163Total 1000 105 1000 105 10000 218 10000 19088

40302010

0

()

Human CT-X genes ()

40302010

0

()

Human autosomal CT genes ()

40302010

0

()

All human CT genes ()

Euka

ryot

a

Opi

sthok

onta

Bila

teria

Coelo

mat

a

Eute

leosto

mi

Amni

ota

Euth

eria

Euar

chon

togl

Cata

rrhi

ni

Hom

inin

ae

Hom

o

40302010

0

()

All annotated human genes ()

Figure 1 The proportions of CT-X genes autosomal CT genes all human CT genes and all annotated human genes with orthologuesoriginated in different taxa of H sapiens lineage

or humans Thus the majority of human CT genes (7248)originated during or after the emergence of Eutheria On theother side the majority of annotated human genes (7595)were older than Eutheria

A significance of the difference between distribution of allhuman genes and all humanCT genes according to the originof their orthologues in different taxa was confirmed bychisquare test (119875 value less than 10minus6)Moreover 95 confidence

region for the cumulative distribution function of CT humangenes displays that CT genes are stochastically younger ascompared to all human genes In other words the probabilitythat a gene randomly chosen from all human genes is youngerthan some fixed time 119879 is less than the probability that arandomly chosen CT gene is younger than119879Therefore thereis a significant bias in time of origin for human CT genes ascompared to all human genes If human CT genes would be

4 International Journal of Genomics

Euka

ryot

a

Opi

sthok

onta

Bila

teria

Coelo

mat

a

Eute

leosto

mi

Amni

ota

Euth

eria

Euar

chon

togli

res

Cata

rrhi

ni

Hom

o sa

pien

s

Hom

inin

ae

1

08

06

04

02

0

cdf for all human genesecdf for studied CT genes95 confidence band

Figure 2 Cumulative distribution function for all human genesand empirical distribution function for all CT human genes inaccordance with the origin of their orthologues in different taxawith 95 confidence bands cdfmdashcumulative distribution func-tion ecdfmdashempirical cumulative distribution function

obtained as a sample from some probabilistic distributionthe probability that CT human genes originated not earlierthan Catarrhini or Eutheria would be significantly higherthan the respective probability for census of all human genes(Figure 2) This statistical trial confirms that the origin ofhuman CT genes is relatively recent

4 Conclusion

Cancertestis antigen genes (CTA or CT genes) encode asubgroup of tumor antigens expressed predominantly intestis and various tumors CT antigens may be also expressedin placenta and in female germ cells [17ndash20] In additionsome CT antigens are expressed in the brain [21]

Experimentally human CT genes were discovered bya variety of immunological screening methods [22] sero-logical identification of antigens by recombinant expressioncloning (SEREX) [23] expression database analysis [2425] massively parallel signature sequencing [26] and otherapproaches The fact that many CT antigens have beenidentified using SEREX suggests that they are highly antigenic[23 27]

The first CT gene discovered was MAGEA1 that encodesfor an antigen of human melanoma [22] This gene belongsto a family of 12 closely related genes clustered at Xq28 Asecond cluster of MAGE genes MAGEB was discovered atXp213 and the third encoding MAGEC genes is located atXq26-27The expression ofMAGEA-MAGEC genes (MAGE-Isubfamily) is restricted to testis and cancer whereasmore dis-tantly related clusters MAGED-MAGEL (subfamily MAGE-II) are expressed in many normal tissuesMAGE-I genes areof relatively recent origin and MAGE-II genes are relativelymore ancient For example MAGE-D genes are conservedbetween man and mouse One of these genes corresponds

to the founder member of the family and the other MAGEgenes are retrogenes derived from the commonancestral gene[19 28 29]

To date CTD atabase (httpwwwctalnccbr) includes265CT genes More than half of them are located on X-chromosome (CT-X genes) [21] The analysis of the DNAsequence of the human X chromosome predicts that approx-imately 10 of the genes on the X chromosome are ofthe CT antigen type [30] Non-X CT genes are distributedthroughout the genome and are representedmainly by single-copy genes [19 27 31]

In normal testis CT-X genes are expressed in prolif-erating germ cells (spermatogonia) Non-X CT genes areexpressed during later stages of germ-cell differentiation thatis spermatocytes [19] Among human tumors CT antigensare expressed in melanoma bladder cancer lung cancerbreast cancer prostate cancer sarcoma ovarian cancer hepa-tocellular carcinoma hematologic malignancies and so forth[21 27 31 32] Genome-wide analysis of 153 cancertestisgenes expression has led to their classification into testis-restricted (119873 = 39) testisbrain-restricted (119873 = 14) andtestis-selective (119873 = 85) groups of genes the latter groupshowing some expression in nongermline tissuesThemajor-ity of testis-restricted genes belong to CT-X group (35 oftotal 39 testis-restricted groups) while non-X CT genes areexpressed in a less restrictive way [21]

Multiple CT antigens are often coexpressed in tumorssuggesting that this expression program is coordinated forentire family [19 23 33] CT gene expression is controlledby epigenetic mechanisms which include DNA methyla-tion and histone posttranslational modifications [31] Othermechanisms of CT gene regulation include sequence-specifictranscription factors and signal transduction pathways suchas activated tyrosine kinases [34]

The functions of CT-X genes are largely unknown Onthe contrary more is known about functions of non-X CTgenes which are associated with meiosis gametogenesis andfertilization Non-X CTs are also more conserved duringevolution [21 27 31 32]

CT-X genes tend to form recently expanded gene familiesmany with nearly identical gene copies [17ndash20 26 32 35]

The prevalence of large highly homologous invertedrepeats (IRs) containing testes genes on the X- and Y-chromosomes was described in humans and great apes [3637] CT-X gene families are also located in direct or invertedrepeats [20]

The study of clusters of homologous genes originated bygene duplication roughly after the divergence of the humanand rodent lineages discovered several families of CT genesamong recent duplicates [38]

In the other paper the authors also studied recentduplications in the human genome and found that CTgenes were represented in this gene set including the familyof PRAME (preferentially expressed antigen of melanoma)genes located on chromosome 1 and expressed in the testisand in a large number of tumors [39] Duplicated PRAMEgenes are hominid specific having arisen in human genomesince the divergence from chimps PRAME gene familyalso expanded in other Eutheria Chimp and mouse have

International Journal of Genomics 5

orthologous PRAME gene clusters on their chromosomes 1and 4 respectively [39 40]

Rapid evolution of cancertestis genes has been demon-strated on the X chromosome In particular the comparisonof human chimp orthologues of these genes has shown thatthey diverge faster and undergo stronger positive selectionthan those on the autosomes or than control genes on eitherX chromosome or autosomes [41]

SPANX-AD gene subfamily of cancertestis-specific anti-gens evolved in the common ancestor of the hominoidlineage after its separation from orangutan Southern blotand database analyses have detected SPANX sequences onlyin primates [17] The coding sequences of the SPANXgenes evolved rapidly faster than their introns and the51015840 untranslated regions with accelerated rates of substi-tutions in both synonymous and nonsynonymous codonpositions The mechanism of SPANX genes expansion wassegmental DNA duplications with evidence of positiveselection SPANX-N is the ancestral form from which theSPANX-AD subfamily evolved in the common ancestor ofhominoids approximately 7 MYA [35 42] SPANX genesare expressed in cancer cells and highly metastatic celllines from melanomas bladder carcinomas and myelomas[35]

The GAGE cancertestis antigen gene family containsat least 16 genes which are encoded by an equal numberof tandem repeats All GAGE genes are located at Xp1123GAGE genes are highly identical and evolved under positiveselection that supports their recent origin [43 44]

The XAGE family of cancertestis antigen genes belongsto superfamily of GAGE-like CT genes It is located on chro-mosomeXp1121-Xp1122ThreeXAGE genes are described aswell as several splice variants of XAGE-1 [45 46]

CT45 gene family was discovered by massively parallelsignature sequencing It includes six highly similar (gt98)genes that are cluctered in tandem on chromosome Xq263CT45 antigen is expressed in Hodgkinrsquos lymphoma and inother human tumors [26 47ndash49]

CT47 cancertestis gene family is located on chromosomeXq24 Among normal tissues it is expressed in the testis and(weakly) in placenta and brain In tumors its expression wasfound in lung cancer and esophageal cancerTheCT47 familymember is characterized by high (gt98) sequence homologyChimp is the only other species in which a gene homologousto CT47 was found by other authors [20]

Our work is the first systematic study of the evolutionarynovelty of the whole class of CT genes To assess the evolu-tionary novelty of CT genes we applied the HomoloGenetool of NCBI To construct the clusters of orthologuesthe HomoloGene program uses information from blastpphylogenetic analyses and syntheny information when it ispossible Cutoffs on bits per position and Ks values are setto prevent unlikely ldquoorthologsrdquo from being grouped togetherThese cutoffs are calculated based on the respective scoredistribution for the given groups of organisms [12]

We searched for orthologues of each of CT genes amongannotated genes in several completely sequenced eukaryoticgenomes and built distributions of all CT-X genes all autoso-mal CT genes all human CT genes and all annotated protein

coding genes from human genome according to the origin oftheir orthologues in 11 taxa of human lineage

We have shown that 314 of CT-X genes are exclusive forhumans and 391ofCT-Xgenes have orthologues originatedin Catarrhini or Homininae Thereby the majority of humanCT-X genes (705) are novel or recent in its origin Our dataare in good correspondence with evidence obtained by othergroups on rapid expansion of certain CT-X gene families andhigh homology of their members which suggest their recentorigin

Altogether 367 of all human CT genes originated inCatarrhini Homininae and humansWe have also found that3073 of all human CT genes originated in Eutheria TheseCT genes acquired functions in Eutheria This indicates theimportance of processes in which tumors and CT antigenswere involved during the evolution of Eutheria CT genesoriginated in Eutheria are located mostly on autosomes CTgenes originated in Catarrhini Homininae and humans arelocated predominantly on X chromosome This difference isprobably related to evolution of mammalian X chromosomesince the origin of Eutheria [50] especially to the acquisitionof its special role in the origin of novel genes [9]

Thus the majority of CT-X genes are either novel oryoung for humans and the majority of all human CT genes(7248) originated during or after the origin of EutheriaThese results suggest that the whole class of human CT genesis relatively evolutionarily new

In its turn this conclusion confirms our prediction aboutexpression of evolutionary recent and novel genes in tumors[10] The expression of cancertestis genes in tumors is thena natural phenomenon not aberrant process as suggested bymany authors (eg [19 27 32 34 40])

References

[1] E E Schmidt ldquoTranscriptional promiscuity in testesrdquo CurrentBiology vol 6 no 7 pp 768ndash769 1996

[2] K C Kleene ldquoSexual selection genetic conflict selfish genesand the atypical patterns of gene expression in spermatogeniccellsrdquo Developmental Biology vol 277 no 1 pp 16ndash26 2005

[3] E Betran K Thornton and M Long ldquoRetroposed new genesout of the X in DrosophilardquoGenome Research vol 12 no 12 pp1854ndash1859 2002

[4] C A Paulding M Ruvolo and D A Haber ldquoThe Tre2(USP6) oncogene is a hominoid-specific generdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 5 pp 2507ndash2511 2003

[5] X She Z Cheng S Zollner D M Church and E E EichlerldquoMouse segmental duplication and copy number variationrdquoNature Genetics vol 40 no 7 pp 909ndash914 2008

[6] M T Levine C D Jones A D Kern H A Lindfors andD J Begun ldquoNovel genes derived from noncoding DNA inDrosophila melanogaster are frequently X-linked and exhibittestis-biased expressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 103 no 26 pp9935ndash9939 2006

[7] T J A J Heinen F Staubach D Haming and D Tautz ldquoEmer-gence of a new gene from an intergenic regionrdquoCurrent Biologyvol 19 no 18 pp 1527ndash1531 2009

6 International Journal of Genomics

[8] H Kaessmann N Vinckenbosch and M Long ldquoRNA-based gene duplication mechanistic and evolutionary insightsrdquoNature Reviews Genetics vol 10 no 1 pp 19ndash31 2009

[9] H Kaessmann ldquoOrigins evolution and phenotypic impact ofnew genesrdquo Genome Research vol 20 no 10 pp 1313ndash13262010

[10] A P Kozlov ldquoThe possible evolutionary role of tumors in theorigin of new cell typesrdquo Medical Hypotheses vol 74 no 1 pp177ndash185 2010

[11] J J Emerson H Kaessmann E Betran and M Long ldquoExten-sive gene traffic on the mammalian X chromosomerdquo Sciencevol 303 no 5657 pp 537ndash540 2004

[12] EW Sayers T Barrett D A Benson et al ldquoDatabase resourcesof the National Center for Biotechnology Informationrdquo NucleicAcids Research vol 40 pp D13ndashD25 2012

[13] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg

[14] H SheffeThe Analysis of Variance Wiley New York NY USA1959

[15] H Sheffe ldquoMultiple testing versus multiple estimationImproper confidence sets Estimation of directions and ratiosrdquoThe Annals of Mathematical Statistics vol 41 no 1 pp 1ndash291970

[16] L A Goodman ldquoSimultaneous confidence intervals for con-trasts among multinomial populationsrdquo The Annals of Mathe-matical Statistics vol 35 pp 716ndash725 1964

[17] A J W Zendman J Zschocke A A Van Kraats et alldquoThe human SPANX multigene family genomic organizationalignment and expression in male germ cells and tumor celllinesrdquo Gene vol 309 no 2 pp 125ndash133 2003

[18] A J W Zendman D J Ruiter and G N P Van MuijenldquoCancertestis-associated genes identification expression pro-file and putative functionrdquo Journal of Cellular Physiology vol194 no 3 pp 272ndash288 2003

[19] A J G Simpson O L Caballero A Jungbluth Y T Chen andL J Old ldquoCancertestis antigens gametogenesis and cancerrdquoNature Reviews Cancer vol 5 no 8 pp 615ndash625 2005

[20] Y T Chen C Iseli C A Yenditti L J Old A J G Simpsonand C V Jongeneel ldquoIdentification of a new cancertestis genefamily CT47 among expressed multicopy genes on the humanX chromosomerdquoGenes Chromosomes and Cancer vol 45 no 4pp 392ndash400 2006

[21] O Hofmann O L Caballero B J Stevenson et al ldquoGenome-wide analysis of cancertestis gene expressionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 51 pp 20422ndash20427 2008

[22] P Van Der Bruggen C Traversari P Chomez et al ldquoA geneencoding an antigen recognized by cytolytic T lymphocytes ona human melanomardquo Science vol 254 no 5038 pp 1643ndash16471991

[23] U Sahin O Tereci H Schmitt et al ldquoHuman neoplasms elicitmultiple specific immune responses in the autologous hostrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11810ndash11813 1995

[24] M J ScanlanCMGordon BWilliamson et al ldquoIdentificationof cancertestis genes by database mining and mRNA expres-sion analysisrdquo International Journal of Cancer vol 98 no 4 pp485ndash492 2002

[25] M J Scanlan A J Simpson and L J Old ldquoThe cancertestisgenes review standardization and commentaryrdquo Cancer Im-munity vol 4 article 1 2004

[26] Y T Chen M J Scanlan C A Venditti et al ldquoIdentificationof cancertestis-antigen genes by massively parallel signaturesequencingrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 102 no 22 pp 7940ndash79452005

[27] Y H Cheng E W P Wong and C Y Cheng ldquoCancertestis(CT) antigens carcinogenesis and spermatogenesisrdquo Spermato-genesis vol 1 pp 209ndash220 2011

[28] P Chomez O De Backer M Bertrand E De Plaen T Boonand S Lucas ldquoAn overview of the MAGE gene family withthe identification of all human members of the familyrdquo CancerResearch vol 61 no 14 pp 5544ndash5551 2001

[29] Y Katsura and Y Satta ldquoEvolutionary history of the cancer Im-munity antigen MAGE gene familyrdquo PLoS ONE vol 6 no 6Article ID e20365 2011

[30] M T Ross D V Grafham A J Coffey et al ldquoThe DNAsequence of the human X chromosomerdquo Nature vol 434 pp325ndash337 2005

[31] E Fratta S Coral A Covre et al ldquoThe biology of cancertestis antigens putative function regulation and therapeuticpotentialrdquoMolecular Oncology vol 5 no 2 pp 164ndash182 2011

[32] O L Caballero and Y T Chen ldquoCancertestis (CT) antigenspotential targets for immunotherapyrdquo Cancer Science vol 100no 11 pp 2014ndash2021 2009

[33] U Sahin O Tureci Y T Chen et al ldquoExpression of multiplecancertestis antigens in breast cancer and melanoma basisfor polyvalent CT vaccine strategiesrdquo International Journal ofCancer vol 78 pp 387ndash389 1998

[34] S N Akers K Odunsi and A R Karpf ldquoRegulation of cancergermline antigen gene expression implications for cancerimmunotherapyrdquo Future Oncology vol 6 no 5 pp 717ndash7322010

[35] N Kouprina MMullokandov I B Rogozin et al ldquoThe SPANXgene family of cancertestis-specific antigens rapid evolutionand amplification in African great apes and hominidsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 9 pp 3077ndash3082 2004

[36] H Skaletsky T Kuroda-Kawaguchl P J Minx et al ldquoThemale-specific region of the human Y chromosome is a mosaic ofdiscrete sequence classesrdquo Nature vol 423 no 6942 pp 825ndash837 2003

[37] P E Warburton J Giordano F Cheung Y Gelfand and GBenson ldquoInverted repeat structure of the humangenome theX-chromosome contains a preponderance of large highly homol-ogous inverted repeated that contain testes genesrdquo GenomeResearch A vol 14 no 10 pp 1861ndash1869 2004

[38] IHGSC ldquoInternational human genome sequencing consor-tiumrdquo Nature vol 431 pp 931ndash945 2004

[39] Z Birtle L Goodstadt and C Ponting ldquoDuplication andpositive selection among hominin-specific PRAME genesrdquoBMC Genomics vol 6 article 120 2005

[40] T C Chang Y Yang H Yasue A K Bharti E F Retzel andWS Liu ldquoThe expansion of the PRAME gene family in EutheriardquoPLoS ONE vol 6 no 2 Article ID e16867 2011

[41] B J Stevenson C Iseli S Panji et al ldquoRapid evolution ofcancertestis genes on the X chromosomerdquo BMCGenomics vol8 article 129 2007

[42] N Kouprina V N Noskov A Pavlicek et al ldquoEvolutionarydiversification of SPANX-N sperm protein gene structure andexpressionrdquo PLoS ONE vol 2 no 4 article e359 2007

International Journal of Genomics 7

[43] Y Liu Q Zhu and N Zhu ldquoRecent duplication and positiveselection of the GAGE gene familyrdquo Genetica vol 133 no 1 pp31ndash35 2008

[44] M F Gjerstorff andH J Ditzel ldquoAn overview of the GAGE can-certestis antigen family with the inclusion of newly identifiedmembersrdquo Tissue Antigens vol 71 no 3 pp 187ndash192 2008

[45] A J W Zendman A A Van Kraats U H Weidle D J Ruiterand G N P Van Muijen ldquoThe XAGE family of cancertestis-associated genes alignment and expression profile in normaltissues melanoma lesions and Ewingrsquos sarcomardquo InternationalJournal of Cancer vol 99 no 3 pp 361ndash369 2002

[46] S Sato Y Noguchi N Ohara et al ldquoIdentification of XAGE-1 isoforms predominant expression of XAGE-1b in testis andtumorsrdquo Cancer Immunity vol 7 article 5 2007

[47] Y T Chen M Hsu P Lee et al ldquoCancertestis antigen CT45analysis of mRNA and protein expression in human cancerrdquoInternational Journal of Cancer vol 124 no 12 pp 2893ndash28982009

[48] Y T Chen A Chadburn P Lee et al ldquoExpression of cancertestis antigen CT45 in classical Hodgkin lymphoma and otherB-cell lymphomasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 7 pp 3093ndash3098 2010

[49] H J Heidebrecht A Claviez M L Kruse et al ldquoCharacteriza-tion and expression of CT45 in Hodgkinrsquos lymphomardquo ClinicalCancer Research vol 12 pp 4804ndash4811 2006

[50] B T Lahn and D C Page ldquoFour evolutionary strata on thehumanX chromosomerdquo Science vol 286 no 5441 pp 964ndash9671999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Genomics 3

Table 2 CT-X genes autosomal CT genes all human CT genes and all annotated human genes with orthologues originated in different taxaof H sapiens lineage

Taxa CT-X genes Autosome CT genes All CT genes All human genesEukaryota 10 1 76 8 413 9 1519 2900Opisthokonta 19 2 092 2 321 613Bilateria 19 2 48 5 321 7 812 1549Coelomata 57 6 275 6 827 1579Euteleostomi 29 3 248 26 1330 29 3277 6256Amniota 67 7 321 7 839 1601Eutheria 200 21 362 38 3073 67 1641 3132Euarchontoglires 38 4 67 7 505 11 175 334Catarrhini 267 28 29 3 1422 31 266 507Homininae 124 13 19 2 688 15 238 454Homo sapiens 314 33 10 1 1560 34 085 163Total 1000 105 1000 105 10000 218 10000 19088

40302010

0

()

Human CT-X genes ()

40302010

0

()

Human autosomal CT genes ()

40302010

0

()

All human CT genes ()

Euka

ryot

a

Opi

sthok

onta

Bila

teria

Coelo

mat

a

Eute

leosto

mi

Amni

ota

Euth

eria

Euar

chon

togl

Cata

rrhi

ni

Hom

inin

ae

Hom

o

40302010

0

()

All annotated human genes ()

Figure 1 The proportions of CT-X genes autosomal CT genes all human CT genes and all annotated human genes with orthologuesoriginated in different taxa of H sapiens lineage

or humans Thus the majority of human CT genes (7248)originated during or after the emergence of Eutheria On theother side the majority of annotated human genes (7595)were older than Eutheria

A significance of the difference between distribution of allhuman genes and all humanCT genes according to the originof their orthologues in different taxa was confirmed bychisquare test (119875 value less than 10minus6)Moreover 95 confidence

region for the cumulative distribution function of CT humangenes displays that CT genes are stochastically younger ascompared to all human genes In other words the probabilitythat a gene randomly chosen from all human genes is youngerthan some fixed time 119879 is less than the probability that arandomly chosen CT gene is younger than119879Therefore thereis a significant bias in time of origin for human CT genes ascompared to all human genes If human CT genes would be

4 International Journal of Genomics

Euka

ryot

a

Opi

sthok

onta

Bila

teria

Coelo

mat

a

Eute

leosto

mi

Amni

ota

Euth

eria

Euar

chon

togli

res

Cata

rrhi

ni

Hom

o sa

pien

s

Hom

inin

ae

1

08

06

04

02

0

cdf for all human genesecdf for studied CT genes95 confidence band

Figure 2 Cumulative distribution function for all human genesand empirical distribution function for all CT human genes inaccordance with the origin of their orthologues in different taxawith 95 confidence bands cdfmdashcumulative distribution func-tion ecdfmdashempirical cumulative distribution function

obtained as a sample from some probabilistic distributionthe probability that CT human genes originated not earlierthan Catarrhini or Eutheria would be significantly higherthan the respective probability for census of all human genes(Figure 2) This statistical trial confirms that the origin ofhuman CT genes is relatively recent

4 Conclusion

Cancertestis antigen genes (CTA or CT genes) encode asubgroup of tumor antigens expressed predominantly intestis and various tumors CT antigens may be also expressedin placenta and in female germ cells [17ndash20] In additionsome CT antigens are expressed in the brain [21]

Experimentally human CT genes were discovered bya variety of immunological screening methods [22] sero-logical identification of antigens by recombinant expressioncloning (SEREX) [23] expression database analysis [2425] massively parallel signature sequencing [26] and otherapproaches The fact that many CT antigens have beenidentified using SEREX suggests that they are highly antigenic[23 27]

The first CT gene discovered was MAGEA1 that encodesfor an antigen of human melanoma [22] This gene belongsto a family of 12 closely related genes clustered at Xq28 Asecond cluster of MAGE genes MAGEB was discovered atXp213 and the third encoding MAGEC genes is located atXq26-27The expression ofMAGEA-MAGEC genes (MAGE-Isubfamily) is restricted to testis and cancer whereasmore dis-tantly related clusters MAGED-MAGEL (subfamily MAGE-II) are expressed in many normal tissuesMAGE-I genes areof relatively recent origin and MAGE-II genes are relativelymore ancient For example MAGE-D genes are conservedbetween man and mouse One of these genes corresponds

to the founder member of the family and the other MAGEgenes are retrogenes derived from the commonancestral gene[19 28 29]

To date CTD atabase (httpwwwctalnccbr) includes265CT genes More than half of them are located on X-chromosome (CT-X genes) [21] The analysis of the DNAsequence of the human X chromosome predicts that approx-imately 10 of the genes on the X chromosome are ofthe CT antigen type [30] Non-X CT genes are distributedthroughout the genome and are representedmainly by single-copy genes [19 27 31]

In normal testis CT-X genes are expressed in prolif-erating germ cells (spermatogonia) Non-X CT genes areexpressed during later stages of germ-cell differentiation thatis spermatocytes [19] Among human tumors CT antigensare expressed in melanoma bladder cancer lung cancerbreast cancer prostate cancer sarcoma ovarian cancer hepa-tocellular carcinoma hematologic malignancies and so forth[21 27 31 32] Genome-wide analysis of 153 cancertestisgenes expression has led to their classification into testis-restricted (119873 = 39) testisbrain-restricted (119873 = 14) andtestis-selective (119873 = 85) groups of genes the latter groupshowing some expression in nongermline tissuesThemajor-ity of testis-restricted genes belong to CT-X group (35 oftotal 39 testis-restricted groups) while non-X CT genes areexpressed in a less restrictive way [21]

Multiple CT antigens are often coexpressed in tumorssuggesting that this expression program is coordinated forentire family [19 23 33] CT gene expression is controlledby epigenetic mechanisms which include DNA methyla-tion and histone posttranslational modifications [31] Othermechanisms of CT gene regulation include sequence-specifictranscription factors and signal transduction pathways suchas activated tyrosine kinases [34]

The functions of CT-X genes are largely unknown Onthe contrary more is known about functions of non-X CTgenes which are associated with meiosis gametogenesis andfertilization Non-X CTs are also more conserved duringevolution [21 27 31 32]

CT-X genes tend to form recently expanded gene familiesmany with nearly identical gene copies [17ndash20 26 32 35]

The prevalence of large highly homologous invertedrepeats (IRs) containing testes genes on the X- and Y-chromosomes was described in humans and great apes [3637] CT-X gene families are also located in direct or invertedrepeats [20]

The study of clusters of homologous genes originated bygene duplication roughly after the divergence of the humanand rodent lineages discovered several families of CT genesamong recent duplicates [38]

In the other paper the authors also studied recentduplications in the human genome and found that CTgenes were represented in this gene set including the familyof PRAME (preferentially expressed antigen of melanoma)genes located on chromosome 1 and expressed in the testisand in a large number of tumors [39] Duplicated PRAMEgenes are hominid specific having arisen in human genomesince the divergence from chimps PRAME gene familyalso expanded in other Eutheria Chimp and mouse have

International Journal of Genomics 5

orthologous PRAME gene clusters on their chromosomes 1and 4 respectively [39 40]

Rapid evolution of cancertestis genes has been demon-strated on the X chromosome In particular the comparisonof human chimp orthologues of these genes has shown thatthey diverge faster and undergo stronger positive selectionthan those on the autosomes or than control genes on eitherX chromosome or autosomes [41]

SPANX-AD gene subfamily of cancertestis-specific anti-gens evolved in the common ancestor of the hominoidlineage after its separation from orangutan Southern blotand database analyses have detected SPANX sequences onlyin primates [17] The coding sequences of the SPANXgenes evolved rapidly faster than their introns and the51015840 untranslated regions with accelerated rates of substi-tutions in both synonymous and nonsynonymous codonpositions The mechanism of SPANX genes expansion wassegmental DNA duplications with evidence of positiveselection SPANX-N is the ancestral form from which theSPANX-AD subfamily evolved in the common ancestor ofhominoids approximately 7 MYA [35 42] SPANX genesare expressed in cancer cells and highly metastatic celllines from melanomas bladder carcinomas and myelomas[35]

The GAGE cancertestis antigen gene family containsat least 16 genes which are encoded by an equal numberof tandem repeats All GAGE genes are located at Xp1123GAGE genes are highly identical and evolved under positiveselection that supports their recent origin [43 44]

The XAGE family of cancertestis antigen genes belongsto superfamily of GAGE-like CT genes It is located on chro-mosomeXp1121-Xp1122ThreeXAGE genes are described aswell as several splice variants of XAGE-1 [45 46]

CT45 gene family was discovered by massively parallelsignature sequencing It includes six highly similar (gt98)genes that are cluctered in tandem on chromosome Xq263CT45 antigen is expressed in Hodgkinrsquos lymphoma and inother human tumors [26 47ndash49]

CT47 cancertestis gene family is located on chromosomeXq24 Among normal tissues it is expressed in the testis and(weakly) in placenta and brain In tumors its expression wasfound in lung cancer and esophageal cancerTheCT47 familymember is characterized by high (gt98) sequence homologyChimp is the only other species in which a gene homologousto CT47 was found by other authors [20]

Our work is the first systematic study of the evolutionarynovelty of the whole class of CT genes To assess the evolu-tionary novelty of CT genes we applied the HomoloGenetool of NCBI To construct the clusters of orthologuesthe HomoloGene program uses information from blastpphylogenetic analyses and syntheny information when it ispossible Cutoffs on bits per position and Ks values are setto prevent unlikely ldquoorthologsrdquo from being grouped togetherThese cutoffs are calculated based on the respective scoredistribution for the given groups of organisms [12]

We searched for orthologues of each of CT genes amongannotated genes in several completely sequenced eukaryoticgenomes and built distributions of all CT-X genes all autoso-mal CT genes all human CT genes and all annotated protein

coding genes from human genome according to the origin oftheir orthologues in 11 taxa of human lineage

We have shown that 314 of CT-X genes are exclusive forhumans and 391ofCT-Xgenes have orthologues originatedin Catarrhini or Homininae Thereby the majority of humanCT-X genes (705) are novel or recent in its origin Our dataare in good correspondence with evidence obtained by othergroups on rapid expansion of certain CT-X gene families andhigh homology of their members which suggest their recentorigin

Altogether 367 of all human CT genes originated inCatarrhini Homininae and humansWe have also found that3073 of all human CT genes originated in Eutheria TheseCT genes acquired functions in Eutheria This indicates theimportance of processes in which tumors and CT antigenswere involved during the evolution of Eutheria CT genesoriginated in Eutheria are located mostly on autosomes CTgenes originated in Catarrhini Homininae and humans arelocated predominantly on X chromosome This difference isprobably related to evolution of mammalian X chromosomesince the origin of Eutheria [50] especially to the acquisitionof its special role in the origin of novel genes [9]

Thus the majority of CT-X genes are either novel oryoung for humans and the majority of all human CT genes(7248) originated during or after the origin of EutheriaThese results suggest that the whole class of human CT genesis relatively evolutionarily new

In its turn this conclusion confirms our prediction aboutexpression of evolutionary recent and novel genes in tumors[10] The expression of cancertestis genes in tumors is thena natural phenomenon not aberrant process as suggested bymany authors (eg [19 27 32 34 40])

References

[1] E E Schmidt ldquoTranscriptional promiscuity in testesrdquo CurrentBiology vol 6 no 7 pp 768ndash769 1996

[2] K C Kleene ldquoSexual selection genetic conflict selfish genesand the atypical patterns of gene expression in spermatogeniccellsrdquo Developmental Biology vol 277 no 1 pp 16ndash26 2005

[3] E Betran K Thornton and M Long ldquoRetroposed new genesout of the X in DrosophilardquoGenome Research vol 12 no 12 pp1854ndash1859 2002

[4] C A Paulding M Ruvolo and D A Haber ldquoThe Tre2(USP6) oncogene is a hominoid-specific generdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 5 pp 2507ndash2511 2003

[5] X She Z Cheng S Zollner D M Church and E E EichlerldquoMouse segmental duplication and copy number variationrdquoNature Genetics vol 40 no 7 pp 909ndash914 2008

[6] M T Levine C D Jones A D Kern H A Lindfors andD J Begun ldquoNovel genes derived from noncoding DNA inDrosophila melanogaster are frequently X-linked and exhibittestis-biased expressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 103 no 26 pp9935ndash9939 2006

[7] T J A J Heinen F Staubach D Haming and D Tautz ldquoEmer-gence of a new gene from an intergenic regionrdquoCurrent Biologyvol 19 no 18 pp 1527ndash1531 2009

6 International Journal of Genomics

[8] H Kaessmann N Vinckenbosch and M Long ldquoRNA-based gene duplication mechanistic and evolutionary insightsrdquoNature Reviews Genetics vol 10 no 1 pp 19ndash31 2009

[9] H Kaessmann ldquoOrigins evolution and phenotypic impact ofnew genesrdquo Genome Research vol 20 no 10 pp 1313ndash13262010

[10] A P Kozlov ldquoThe possible evolutionary role of tumors in theorigin of new cell typesrdquo Medical Hypotheses vol 74 no 1 pp177ndash185 2010

[11] J J Emerson H Kaessmann E Betran and M Long ldquoExten-sive gene traffic on the mammalian X chromosomerdquo Sciencevol 303 no 5657 pp 537ndash540 2004

[12] EW Sayers T Barrett D A Benson et al ldquoDatabase resourcesof the National Center for Biotechnology Informationrdquo NucleicAcids Research vol 40 pp D13ndashD25 2012

[13] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg

[14] H SheffeThe Analysis of Variance Wiley New York NY USA1959

[15] H Sheffe ldquoMultiple testing versus multiple estimationImproper confidence sets Estimation of directions and ratiosrdquoThe Annals of Mathematical Statistics vol 41 no 1 pp 1ndash291970

[16] L A Goodman ldquoSimultaneous confidence intervals for con-trasts among multinomial populationsrdquo The Annals of Mathe-matical Statistics vol 35 pp 716ndash725 1964

[17] A J W Zendman J Zschocke A A Van Kraats et alldquoThe human SPANX multigene family genomic organizationalignment and expression in male germ cells and tumor celllinesrdquo Gene vol 309 no 2 pp 125ndash133 2003

[18] A J W Zendman D J Ruiter and G N P Van MuijenldquoCancertestis-associated genes identification expression pro-file and putative functionrdquo Journal of Cellular Physiology vol194 no 3 pp 272ndash288 2003

[19] A J G Simpson O L Caballero A Jungbluth Y T Chen andL J Old ldquoCancertestis antigens gametogenesis and cancerrdquoNature Reviews Cancer vol 5 no 8 pp 615ndash625 2005

[20] Y T Chen C Iseli C A Yenditti L J Old A J G Simpsonand C V Jongeneel ldquoIdentification of a new cancertestis genefamily CT47 among expressed multicopy genes on the humanX chromosomerdquoGenes Chromosomes and Cancer vol 45 no 4pp 392ndash400 2006

[21] O Hofmann O L Caballero B J Stevenson et al ldquoGenome-wide analysis of cancertestis gene expressionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 51 pp 20422ndash20427 2008

[22] P Van Der Bruggen C Traversari P Chomez et al ldquoA geneencoding an antigen recognized by cytolytic T lymphocytes ona human melanomardquo Science vol 254 no 5038 pp 1643ndash16471991

[23] U Sahin O Tereci H Schmitt et al ldquoHuman neoplasms elicitmultiple specific immune responses in the autologous hostrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11810ndash11813 1995

[24] M J ScanlanCMGordon BWilliamson et al ldquoIdentificationof cancertestis genes by database mining and mRNA expres-sion analysisrdquo International Journal of Cancer vol 98 no 4 pp485ndash492 2002

[25] M J Scanlan A J Simpson and L J Old ldquoThe cancertestisgenes review standardization and commentaryrdquo Cancer Im-munity vol 4 article 1 2004

[26] Y T Chen M J Scanlan C A Venditti et al ldquoIdentificationof cancertestis-antigen genes by massively parallel signaturesequencingrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 102 no 22 pp 7940ndash79452005

[27] Y H Cheng E W P Wong and C Y Cheng ldquoCancertestis(CT) antigens carcinogenesis and spermatogenesisrdquo Spermato-genesis vol 1 pp 209ndash220 2011

[28] P Chomez O De Backer M Bertrand E De Plaen T Boonand S Lucas ldquoAn overview of the MAGE gene family withthe identification of all human members of the familyrdquo CancerResearch vol 61 no 14 pp 5544ndash5551 2001

[29] Y Katsura and Y Satta ldquoEvolutionary history of the cancer Im-munity antigen MAGE gene familyrdquo PLoS ONE vol 6 no 6Article ID e20365 2011

[30] M T Ross D V Grafham A J Coffey et al ldquoThe DNAsequence of the human X chromosomerdquo Nature vol 434 pp325ndash337 2005

[31] E Fratta S Coral A Covre et al ldquoThe biology of cancertestis antigens putative function regulation and therapeuticpotentialrdquoMolecular Oncology vol 5 no 2 pp 164ndash182 2011

[32] O L Caballero and Y T Chen ldquoCancertestis (CT) antigenspotential targets for immunotherapyrdquo Cancer Science vol 100no 11 pp 2014ndash2021 2009

[33] U Sahin O Tureci Y T Chen et al ldquoExpression of multiplecancertestis antigens in breast cancer and melanoma basisfor polyvalent CT vaccine strategiesrdquo International Journal ofCancer vol 78 pp 387ndash389 1998

[34] S N Akers K Odunsi and A R Karpf ldquoRegulation of cancergermline antigen gene expression implications for cancerimmunotherapyrdquo Future Oncology vol 6 no 5 pp 717ndash7322010

[35] N Kouprina MMullokandov I B Rogozin et al ldquoThe SPANXgene family of cancertestis-specific antigens rapid evolutionand amplification in African great apes and hominidsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 9 pp 3077ndash3082 2004

[36] H Skaletsky T Kuroda-Kawaguchl P J Minx et al ldquoThemale-specific region of the human Y chromosome is a mosaic ofdiscrete sequence classesrdquo Nature vol 423 no 6942 pp 825ndash837 2003

[37] P E Warburton J Giordano F Cheung Y Gelfand and GBenson ldquoInverted repeat structure of the humangenome theX-chromosome contains a preponderance of large highly homol-ogous inverted repeated that contain testes genesrdquo GenomeResearch A vol 14 no 10 pp 1861ndash1869 2004

[38] IHGSC ldquoInternational human genome sequencing consor-tiumrdquo Nature vol 431 pp 931ndash945 2004

[39] Z Birtle L Goodstadt and C Ponting ldquoDuplication andpositive selection among hominin-specific PRAME genesrdquoBMC Genomics vol 6 article 120 2005

[40] T C Chang Y Yang H Yasue A K Bharti E F Retzel andWS Liu ldquoThe expansion of the PRAME gene family in EutheriardquoPLoS ONE vol 6 no 2 Article ID e16867 2011

[41] B J Stevenson C Iseli S Panji et al ldquoRapid evolution ofcancertestis genes on the X chromosomerdquo BMCGenomics vol8 article 129 2007

[42] N Kouprina V N Noskov A Pavlicek et al ldquoEvolutionarydiversification of SPANX-N sperm protein gene structure andexpressionrdquo PLoS ONE vol 2 no 4 article e359 2007

International Journal of Genomics 7

[43] Y Liu Q Zhu and N Zhu ldquoRecent duplication and positiveselection of the GAGE gene familyrdquo Genetica vol 133 no 1 pp31ndash35 2008

[44] M F Gjerstorff andH J Ditzel ldquoAn overview of the GAGE can-certestis antigen family with the inclusion of newly identifiedmembersrdquo Tissue Antigens vol 71 no 3 pp 187ndash192 2008

[45] A J W Zendman A A Van Kraats U H Weidle D J Ruiterand G N P Van Muijen ldquoThe XAGE family of cancertestis-associated genes alignment and expression profile in normaltissues melanoma lesions and Ewingrsquos sarcomardquo InternationalJournal of Cancer vol 99 no 3 pp 361ndash369 2002

[46] S Sato Y Noguchi N Ohara et al ldquoIdentification of XAGE-1 isoforms predominant expression of XAGE-1b in testis andtumorsrdquo Cancer Immunity vol 7 article 5 2007

[47] Y T Chen M Hsu P Lee et al ldquoCancertestis antigen CT45analysis of mRNA and protein expression in human cancerrdquoInternational Journal of Cancer vol 124 no 12 pp 2893ndash28982009

[48] Y T Chen A Chadburn P Lee et al ldquoExpression of cancertestis antigen CT45 in classical Hodgkin lymphoma and otherB-cell lymphomasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 7 pp 3093ndash3098 2010

[49] H J Heidebrecht A Claviez M L Kruse et al ldquoCharacteriza-tion and expression of CT45 in Hodgkinrsquos lymphomardquo ClinicalCancer Research vol 12 pp 4804ndash4811 2006

[50] B T Lahn and D C Page ldquoFour evolutionary strata on thehumanX chromosomerdquo Science vol 286 no 5441 pp 964ndash9671999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 International Journal of Genomics

Euka

ryot

a

Opi

sthok

onta

Bila

teria

Coelo

mat

a

Eute

leosto

mi

Amni

ota

Euth

eria

Euar

chon

togli

res

Cata

rrhi

ni

Hom

o sa

pien

s

Hom

inin

ae

1

08

06

04

02

0

cdf for all human genesecdf for studied CT genes95 confidence band

Figure 2 Cumulative distribution function for all human genesand empirical distribution function for all CT human genes inaccordance with the origin of their orthologues in different taxawith 95 confidence bands cdfmdashcumulative distribution func-tion ecdfmdashempirical cumulative distribution function

obtained as a sample from some probabilistic distributionthe probability that CT human genes originated not earlierthan Catarrhini or Eutheria would be significantly higherthan the respective probability for census of all human genes(Figure 2) This statistical trial confirms that the origin ofhuman CT genes is relatively recent

4 Conclusion

Cancertestis antigen genes (CTA or CT genes) encode asubgroup of tumor antigens expressed predominantly intestis and various tumors CT antigens may be also expressedin placenta and in female germ cells [17ndash20] In additionsome CT antigens are expressed in the brain [21]

Experimentally human CT genes were discovered bya variety of immunological screening methods [22] sero-logical identification of antigens by recombinant expressioncloning (SEREX) [23] expression database analysis [2425] massively parallel signature sequencing [26] and otherapproaches The fact that many CT antigens have beenidentified using SEREX suggests that they are highly antigenic[23 27]

The first CT gene discovered was MAGEA1 that encodesfor an antigen of human melanoma [22] This gene belongsto a family of 12 closely related genes clustered at Xq28 Asecond cluster of MAGE genes MAGEB was discovered atXp213 and the third encoding MAGEC genes is located atXq26-27The expression ofMAGEA-MAGEC genes (MAGE-Isubfamily) is restricted to testis and cancer whereasmore dis-tantly related clusters MAGED-MAGEL (subfamily MAGE-II) are expressed in many normal tissuesMAGE-I genes areof relatively recent origin and MAGE-II genes are relativelymore ancient For example MAGE-D genes are conservedbetween man and mouse One of these genes corresponds

to the founder member of the family and the other MAGEgenes are retrogenes derived from the commonancestral gene[19 28 29]

To date CTD atabase (httpwwwctalnccbr) includes265CT genes More than half of them are located on X-chromosome (CT-X genes) [21] The analysis of the DNAsequence of the human X chromosome predicts that approx-imately 10 of the genes on the X chromosome are ofthe CT antigen type [30] Non-X CT genes are distributedthroughout the genome and are representedmainly by single-copy genes [19 27 31]

In normal testis CT-X genes are expressed in prolif-erating germ cells (spermatogonia) Non-X CT genes areexpressed during later stages of germ-cell differentiation thatis spermatocytes [19] Among human tumors CT antigensare expressed in melanoma bladder cancer lung cancerbreast cancer prostate cancer sarcoma ovarian cancer hepa-tocellular carcinoma hematologic malignancies and so forth[21 27 31 32] Genome-wide analysis of 153 cancertestisgenes expression has led to their classification into testis-restricted (119873 = 39) testisbrain-restricted (119873 = 14) andtestis-selective (119873 = 85) groups of genes the latter groupshowing some expression in nongermline tissuesThemajor-ity of testis-restricted genes belong to CT-X group (35 oftotal 39 testis-restricted groups) while non-X CT genes areexpressed in a less restrictive way [21]

Multiple CT antigens are often coexpressed in tumorssuggesting that this expression program is coordinated forentire family [19 23 33] CT gene expression is controlledby epigenetic mechanisms which include DNA methyla-tion and histone posttranslational modifications [31] Othermechanisms of CT gene regulation include sequence-specifictranscription factors and signal transduction pathways suchas activated tyrosine kinases [34]

The functions of CT-X genes are largely unknown Onthe contrary more is known about functions of non-X CTgenes which are associated with meiosis gametogenesis andfertilization Non-X CTs are also more conserved duringevolution [21 27 31 32]

CT-X genes tend to form recently expanded gene familiesmany with nearly identical gene copies [17ndash20 26 32 35]

The prevalence of large highly homologous invertedrepeats (IRs) containing testes genes on the X- and Y-chromosomes was described in humans and great apes [3637] CT-X gene families are also located in direct or invertedrepeats [20]

The study of clusters of homologous genes originated bygene duplication roughly after the divergence of the humanand rodent lineages discovered several families of CT genesamong recent duplicates [38]

In the other paper the authors also studied recentduplications in the human genome and found that CTgenes were represented in this gene set including the familyof PRAME (preferentially expressed antigen of melanoma)genes located on chromosome 1 and expressed in the testisand in a large number of tumors [39] Duplicated PRAMEgenes are hominid specific having arisen in human genomesince the divergence from chimps PRAME gene familyalso expanded in other Eutheria Chimp and mouse have

International Journal of Genomics 5

orthologous PRAME gene clusters on their chromosomes 1and 4 respectively [39 40]

Rapid evolution of cancertestis genes has been demon-strated on the X chromosome In particular the comparisonof human chimp orthologues of these genes has shown thatthey diverge faster and undergo stronger positive selectionthan those on the autosomes or than control genes on eitherX chromosome or autosomes [41]

SPANX-AD gene subfamily of cancertestis-specific anti-gens evolved in the common ancestor of the hominoidlineage after its separation from orangutan Southern blotand database analyses have detected SPANX sequences onlyin primates [17] The coding sequences of the SPANXgenes evolved rapidly faster than their introns and the51015840 untranslated regions with accelerated rates of substi-tutions in both synonymous and nonsynonymous codonpositions The mechanism of SPANX genes expansion wassegmental DNA duplications with evidence of positiveselection SPANX-N is the ancestral form from which theSPANX-AD subfamily evolved in the common ancestor ofhominoids approximately 7 MYA [35 42] SPANX genesare expressed in cancer cells and highly metastatic celllines from melanomas bladder carcinomas and myelomas[35]

The GAGE cancertestis antigen gene family containsat least 16 genes which are encoded by an equal numberof tandem repeats All GAGE genes are located at Xp1123GAGE genes are highly identical and evolved under positiveselection that supports their recent origin [43 44]

The XAGE family of cancertestis antigen genes belongsto superfamily of GAGE-like CT genes It is located on chro-mosomeXp1121-Xp1122ThreeXAGE genes are described aswell as several splice variants of XAGE-1 [45 46]

CT45 gene family was discovered by massively parallelsignature sequencing It includes six highly similar (gt98)genes that are cluctered in tandem on chromosome Xq263CT45 antigen is expressed in Hodgkinrsquos lymphoma and inother human tumors [26 47ndash49]

CT47 cancertestis gene family is located on chromosomeXq24 Among normal tissues it is expressed in the testis and(weakly) in placenta and brain In tumors its expression wasfound in lung cancer and esophageal cancerTheCT47 familymember is characterized by high (gt98) sequence homologyChimp is the only other species in which a gene homologousto CT47 was found by other authors [20]

Our work is the first systematic study of the evolutionarynovelty of the whole class of CT genes To assess the evolu-tionary novelty of CT genes we applied the HomoloGenetool of NCBI To construct the clusters of orthologuesthe HomoloGene program uses information from blastpphylogenetic analyses and syntheny information when it ispossible Cutoffs on bits per position and Ks values are setto prevent unlikely ldquoorthologsrdquo from being grouped togetherThese cutoffs are calculated based on the respective scoredistribution for the given groups of organisms [12]

We searched for orthologues of each of CT genes amongannotated genes in several completely sequenced eukaryoticgenomes and built distributions of all CT-X genes all autoso-mal CT genes all human CT genes and all annotated protein

coding genes from human genome according to the origin oftheir orthologues in 11 taxa of human lineage

We have shown that 314 of CT-X genes are exclusive forhumans and 391ofCT-Xgenes have orthologues originatedin Catarrhini or Homininae Thereby the majority of humanCT-X genes (705) are novel or recent in its origin Our dataare in good correspondence with evidence obtained by othergroups on rapid expansion of certain CT-X gene families andhigh homology of their members which suggest their recentorigin

Altogether 367 of all human CT genes originated inCatarrhini Homininae and humansWe have also found that3073 of all human CT genes originated in Eutheria TheseCT genes acquired functions in Eutheria This indicates theimportance of processes in which tumors and CT antigenswere involved during the evolution of Eutheria CT genesoriginated in Eutheria are located mostly on autosomes CTgenes originated in Catarrhini Homininae and humans arelocated predominantly on X chromosome This difference isprobably related to evolution of mammalian X chromosomesince the origin of Eutheria [50] especially to the acquisitionof its special role in the origin of novel genes [9]

Thus the majority of CT-X genes are either novel oryoung for humans and the majority of all human CT genes(7248) originated during or after the origin of EutheriaThese results suggest that the whole class of human CT genesis relatively evolutionarily new

In its turn this conclusion confirms our prediction aboutexpression of evolutionary recent and novel genes in tumors[10] The expression of cancertestis genes in tumors is thena natural phenomenon not aberrant process as suggested bymany authors (eg [19 27 32 34 40])

References

[1] E E Schmidt ldquoTranscriptional promiscuity in testesrdquo CurrentBiology vol 6 no 7 pp 768ndash769 1996

[2] K C Kleene ldquoSexual selection genetic conflict selfish genesand the atypical patterns of gene expression in spermatogeniccellsrdquo Developmental Biology vol 277 no 1 pp 16ndash26 2005

[3] E Betran K Thornton and M Long ldquoRetroposed new genesout of the X in DrosophilardquoGenome Research vol 12 no 12 pp1854ndash1859 2002

[4] C A Paulding M Ruvolo and D A Haber ldquoThe Tre2(USP6) oncogene is a hominoid-specific generdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 5 pp 2507ndash2511 2003

[5] X She Z Cheng S Zollner D M Church and E E EichlerldquoMouse segmental duplication and copy number variationrdquoNature Genetics vol 40 no 7 pp 909ndash914 2008

[6] M T Levine C D Jones A D Kern H A Lindfors andD J Begun ldquoNovel genes derived from noncoding DNA inDrosophila melanogaster are frequently X-linked and exhibittestis-biased expressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 103 no 26 pp9935ndash9939 2006

[7] T J A J Heinen F Staubach D Haming and D Tautz ldquoEmer-gence of a new gene from an intergenic regionrdquoCurrent Biologyvol 19 no 18 pp 1527ndash1531 2009

6 International Journal of Genomics

[8] H Kaessmann N Vinckenbosch and M Long ldquoRNA-based gene duplication mechanistic and evolutionary insightsrdquoNature Reviews Genetics vol 10 no 1 pp 19ndash31 2009

[9] H Kaessmann ldquoOrigins evolution and phenotypic impact ofnew genesrdquo Genome Research vol 20 no 10 pp 1313ndash13262010

[10] A P Kozlov ldquoThe possible evolutionary role of tumors in theorigin of new cell typesrdquo Medical Hypotheses vol 74 no 1 pp177ndash185 2010

[11] J J Emerson H Kaessmann E Betran and M Long ldquoExten-sive gene traffic on the mammalian X chromosomerdquo Sciencevol 303 no 5657 pp 537ndash540 2004

[12] EW Sayers T Barrett D A Benson et al ldquoDatabase resourcesof the National Center for Biotechnology Informationrdquo NucleicAcids Research vol 40 pp D13ndashD25 2012

[13] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg

[14] H SheffeThe Analysis of Variance Wiley New York NY USA1959

[15] H Sheffe ldquoMultiple testing versus multiple estimationImproper confidence sets Estimation of directions and ratiosrdquoThe Annals of Mathematical Statistics vol 41 no 1 pp 1ndash291970

[16] L A Goodman ldquoSimultaneous confidence intervals for con-trasts among multinomial populationsrdquo The Annals of Mathe-matical Statistics vol 35 pp 716ndash725 1964

[17] A J W Zendman J Zschocke A A Van Kraats et alldquoThe human SPANX multigene family genomic organizationalignment and expression in male germ cells and tumor celllinesrdquo Gene vol 309 no 2 pp 125ndash133 2003

[18] A J W Zendman D J Ruiter and G N P Van MuijenldquoCancertestis-associated genes identification expression pro-file and putative functionrdquo Journal of Cellular Physiology vol194 no 3 pp 272ndash288 2003

[19] A J G Simpson O L Caballero A Jungbluth Y T Chen andL J Old ldquoCancertestis antigens gametogenesis and cancerrdquoNature Reviews Cancer vol 5 no 8 pp 615ndash625 2005

[20] Y T Chen C Iseli C A Yenditti L J Old A J G Simpsonand C V Jongeneel ldquoIdentification of a new cancertestis genefamily CT47 among expressed multicopy genes on the humanX chromosomerdquoGenes Chromosomes and Cancer vol 45 no 4pp 392ndash400 2006

[21] O Hofmann O L Caballero B J Stevenson et al ldquoGenome-wide analysis of cancertestis gene expressionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 51 pp 20422ndash20427 2008

[22] P Van Der Bruggen C Traversari P Chomez et al ldquoA geneencoding an antigen recognized by cytolytic T lymphocytes ona human melanomardquo Science vol 254 no 5038 pp 1643ndash16471991

[23] U Sahin O Tereci H Schmitt et al ldquoHuman neoplasms elicitmultiple specific immune responses in the autologous hostrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11810ndash11813 1995

[24] M J ScanlanCMGordon BWilliamson et al ldquoIdentificationof cancertestis genes by database mining and mRNA expres-sion analysisrdquo International Journal of Cancer vol 98 no 4 pp485ndash492 2002

[25] M J Scanlan A J Simpson and L J Old ldquoThe cancertestisgenes review standardization and commentaryrdquo Cancer Im-munity vol 4 article 1 2004

[26] Y T Chen M J Scanlan C A Venditti et al ldquoIdentificationof cancertestis-antigen genes by massively parallel signaturesequencingrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 102 no 22 pp 7940ndash79452005

[27] Y H Cheng E W P Wong and C Y Cheng ldquoCancertestis(CT) antigens carcinogenesis and spermatogenesisrdquo Spermato-genesis vol 1 pp 209ndash220 2011

[28] P Chomez O De Backer M Bertrand E De Plaen T Boonand S Lucas ldquoAn overview of the MAGE gene family withthe identification of all human members of the familyrdquo CancerResearch vol 61 no 14 pp 5544ndash5551 2001

[29] Y Katsura and Y Satta ldquoEvolutionary history of the cancer Im-munity antigen MAGE gene familyrdquo PLoS ONE vol 6 no 6Article ID e20365 2011

[30] M T Ross D V Grafham A J Coffey et al ldquoThe DNAsequence of the human X chromosomerdquo Nature vol 434 pp325ndash337 2005

[31] E Fratta S Coral A Covre et al ldquoThe biology of cancertestis antigens putative function regulation and therapeuticpotentialrdquoMolecular Oncology vol 5 no 2 pp 164ndash182 2011

[32] O L Caballero and Y T Chen ldquoCancertestis (CT) antigenspotential targets for immunotherapyrdquo Cancer Science vol 100no 11 pp 2014ndash2021 2009

[33] U Sahin O Tureci Y T Chen et al ldquoExpression of multiplecancertestis antigens in breast cancer and melanoma basisfor polyvalent CT vaccine strategiesrdquo International Journal ofCancer vol 78 pp 387ndash389 1998

[34] S N Akers K Odunsi and A R Karpf ldquoRegulation of cancergermline antigen gene expression implications for cancerimmunotherapyrdquo Future Oncology vol 6 no 5 pp 717ndash7322010

[35] N Kouprina MMullokandov I B Rogozin et al ldquoThe SPANXgene family of cancertestis-specific antigens rapid evolutionand amplification in African great apes and hominidsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 9 pp 3077ndash3082 2004

[36] H Skaletsky T Kuroda-Kawaguchl P J Minx et al ldquoThemale-specific region of the human Y chromosome is a mosaic ofdiscrete sequence classesrdquo Nature vol 423 no 6942 pp 825ndash837 2003

[37] P E Warburton J Giordano F Cheung Y Gelfand and GBenson ldquoInverted repeat structure of the humangenome theX-chromosome contains a preponderance of large highly homol-ogous inverted repeated that contain testes genesrdquo GenomeResearch A vol 14 no 10 pp 1861ndash1869 2004

[38] IHGSC ldquoInternational human genome sequencing consor-tiumrdquo Nature vol 431 pp 931ndash945 2004

[39] Z Birtle L Goodstadt and C Ponting ldquoDuplication andpositive selection among hominin-specific PRAME genesrdquoBMC Genomics vol 6 article 120 2005

[40] T C Chang Y Yang H Yasue A K Bharti E F Retzel andWS Liu ldquoThe expansion of the PRAME gene family in EutheriardquoPLoS ONE vol 6 no 2 Article ID e16867 2011

[41] B J Stevenson C Iseli S Panji et al ldquoRapid evolution ofcancertestis genes on the X chromosomerdquo BMCGenomics vol8 article 129 2007

[42] N Kouprina V N Noskov A Pavlicek et al ldquoEvolutionarydiversification of SPANX-N sperm protein gene structure andexpressionrdquo PLoS ONE vol 2 no 4 article e359 2007

International Journal of Genomics 7

[43] Y Liu Q Zhu and N Zhu ldquoRecent duplication and positiveselection of the GAGE gene familyrdquo Genetica vol 133 no 1 pp31ndash35 2008

[44] M F Gjerstorff andH J Ditzel ldquoAn overview of the GAGE can-certestis antigen family with the inclusion of newly identifiedmembersrdquo Tissue Antigens vol 71 no 3 pp 187ndash192 2008

[45] A J W Zendman A A Van Kraats U H Weidle D J Ruiterand G N P Van Muijen ldquoThe XAGE family of cancertestis-associated genes alignment and expression profile in normaltissues melanoma lesions and Ewingrsquos sarcomardquo InternationalJournal of Cancer vol 99 no 3 pp 361ndash369 2002

[46] S Sato Y Noguchi N Ohara et al ldquoIdentification of XAGE-1 isoforms predominant expression of XAGE-1b in testis andtumorsrdquo Cancer Immunity vol 7 article 5 2007

[47] Y T Chen M Hsu P Lee et al ldquoCancertestis antigen CT45analysis of mRNA and protein expression in human cancerrdquoInternational Journal of Cancer vol 124 no 12 pp 2893ndash28982009

[48] Y T Chen A Chadburn P Lee et al ldquoExpression of cancertestis antigen CT45 in classical Hodgkin lymphoma and otherB-cell lymphomasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 7 pp 3093ndash3098 2010

[49] H J Heidebrecht A Claviez M L Kruse et al ldquoCharacteriza-tion and expression of CT45 in Hodgkinrsquos lymphomardquo ClinicalCancer Research vol 12 pp 4804ndash4811 2006

[50] B T Lahn and D C Page ldquoFour evolutionary strata on thehumanX chromosomerdquo Science vol 286 no 5441 pp 964ndash9671999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Genomics 5

orthologous PRAME gene clusters on their chromosomes 1and 4 respectively [39 40]

Rapid evolution of cancertestis genes has been demon-strated on the X chromosome In particular the comparisonof human chimp orthologues of these genes has shown thatthey diverge faster and undergo stronger positive selectionthan those on the autosomes or than control genes on eitherX chromosome or autosomes [41]

SPANX-AD gene subfamily of cancertestis-specific anti-gens evolved in the common ancestor of the hominoidlineage after its separation from orangutan Southern blotand database analyses have detected SPANX sequences onlyin primates [17] The coding sequences of the SPANXgenes evolved rapidly faster than their introns and the51015840 untranslated regions with accelerated rates of substi-tutions in both synonymous and nonsynonymous codonpositions The mechanism of SPANX genes expansion wassegmental DNA duplications with evidence of positiveselection SPANX-N is the ancestral form from which theSPANX-AD subfamily evolved in the common ancestor ofhominoids approximately 7 MYA [35 42] SPANX genesare expressed in cancer cells and highly metastatic celllines from melanomas bladder carcinomas and myelomas[35]

The GAGE cancertestis antigen gene family containsat least 16 genes which are encoded by an equal numberof tandem repeats All GAGE genes are located at Xp1123GAGE genes are highly identical and evolved under positiveselection that supports their recent origin [43 44]

The XAGE family of cancertestis antigen genes belongsto superfamily of GAGE-like CT genes It is located on chro-mosomeXp1121-Xp1122ThreeXAGE genes are described aswell as several splice variants of XAGE-1 [45 46]

CT45 gene family was discovered by massively parallelsignature sequencing It includes six highly similar (gt98)genes that are cluctered in tandem on chromosome Xq263CT45 antigen is expressed in Hodgkinrsquos lymphoma and inother human tumors [26 47ndash49]

CT47 cancertestis gene family is located on chromosomeXq24 Among normal tissues it is expressed in the testis and(weakly) in placenta and brain In tumors its expression wasfound in lung cancer and esophageal cancerTheCT47 familymember is characterized by high (gt98) sequence homologyChimp is the only other species in which a gene homologousto CT47 was found by other authors [20]

Our work is the first systematic study of the evolutionarynovelty of the whole class of CT genes To assess the evolu-tionary novelty of CT genes we applied the HomoloGenetool of NCBI To construct the clusters of orthologuesthe HomoloGene program uses information from blastpphylogenetic analyses and syntheny information when it ispossible Cutoffs on bits per position and Ks values are setto prevent unlikely ldquoorthologsrdquo from being grouped togetherThese cutoffs are calculated based on the respective scoredistribution for the given groups of organisms [12]

We searched for orthologues of each of CT genes amongannotated genes in several completely sequenced eukaryoticgenomes and built distributions of all CT-X genes all autoso-mal CT genes all human CT genes and all annotated protein

coding genes from human genome according to the origin oftheir orthologues in 11 taxa of human lineage

We have shown that 314 of CT-X genes are exclusive forhumans and 391ofCT-Xgenes have orthologues originatedin Catarrhini or Homininae Thereby the majority of humanCT-X genes (705) are novel or recent in its origin Our dataare in good correspondence with evidence obtained by othergroups on rapid expansion of certain CT-X gene families andhigh homology of their members which suggest their recentorigin

Altogether 367 of all human CT genes originated inCatarrhini Homininae and humansWe have also found that3073 of all human CT genes originated in Eutheria TheseCT genes acquired functions in Eutheria This indicates theimportance of processes in which tumors and CT antigenswere involved during the evolution of Eutheria CT genesoriginated in Eutheria are located mostly on autosomes CTgenes originated in Catarrhini Homininae and humans arelocated predominantly on X chromosome This difference isprobably related to evolution of mammalian X chromosomesince the origin of Eutheria [50] especially to the acquisitionof its special role in the origin of novel genes [9]

Thus the majority of CT-X genes are either novel oryoung for humans and the majority of all human CT genes(7248) originated during or after the origin of EutheriaThese results suggest that the whole class of human CT genesis relatively evolutionarily new

In its turn this conclusion confirms our prediction aboutexpression of evolutionary recent and novel genes in tumors[10] The expression of cancertestis genes in tumors is thena natural phenomenon not aberrant process as suggested bymany authors (eg [19 27 32 34 40])

References

[1] E E Schmidt ldquoTranscriptional promiscuity in testesrdquo CurrentBiology vol 6 no 7 pp 768ndash769 1996

[2] K C Kleene ldquoSexual selection genetic conflict selfish genesand the atypical patterns of gene expression in spermatogeniccellsrdquo Developmental Biology vol 277 no 1 pp 16ndash26 2005

[3] E Betran K Thornton and M Long ldquoRetroposed new genesout of the X in DrosophilardquoGenome Research vol 12 no 12 pp1854ndash1859 2002

[4] C A Paulding M Ruvolo and D A Haber ldquoThe Tre2(USP6) oncogene is a hominoid-specific generdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 100 no 5 pp 2507ndash2511 2003

[5] X She Z Cheng S Zollner D M Church and E E EichlerldquoMouse segmental duplication and copy number variationrdquoNature Genetics vol 40 no 7 pp 909ndash914 2008

[6] M T Levine C D Jones A D Kern H A Lindfors andD J Begun ldquoNovel genes derived from noncoding DNA inDrosophila melanogaster are frequently X-linked and exhibittestis-biased expressionrdquo Proceedings of the National Academyof Sciences of the United States of America vol 103 no 26 pp9935ndash9939 2006

[7] T J A J Heinen F Staubach D Haming and D Tautz ldquoEmer-gence of a new gene from an intergenic regionrdquoCurrent Biologyvol 19 no 18 pp 1527ndash1531 2009

6 International Journal of Genomics

[8] H Kaessmann N Vinckenbosch and M Long ldquoRNA-based gene duplication mechanistic and evolutionary insightsrdquoNature Reviews Genetics vol 10 no 1 pp 19ndash31 2009

[9] H Kaessmann ldquoOrigins evolution and phenotypic impact ofnew genesrdquo Genome Research vol 20 no 10 pp 1313ndash13262010

[10] A P Kozlov ldquoThe possible evolutionary role of tumors in theorigin of new cell typesrdquo Medical Hypotheses vol 74 no 1 pp177ndash185 2010

[11] J J Emerson H Kaessmann E Betran and M Long ldquoExten-sive gene traffic on the mammalian X chromosomerdquo Sciencevol 303 no 5657 pp 537ndash540 2004

[12] EW Sayers T Barrett D A Benson et al ldquoDatabase resourcesof the National Center for Biotechnology Informationrdquo NucleicAcids Research vol 40 pp D13ndashD25 2012

[13] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg

[14] H SheffeThe Analysis of Variance Wiley New York NY USA1959

[15] H Sheffe ldquoMultiple testing versus multiple estimationImproper confidence sets Estimation of directions and ratiosrdquoThe Annals of Mathematical Statistics vol 41 no 1 pp 1ndash291970

[16] L A Goodman ldquoSimultaneous confidence intervals for con-trasts among multinomial populationsrdquo The Annals of Mathe-matical Statistics vol 35 pp 716ndash725 1964

[17] A J W Zendman J Zschocke A A Van Kraats et alldquoThe human SPANX multigene family genomic organizationalignment and expression in male germ cells and tumor celllinesrdquo Gene vol 309 no 2 pp 125ndash133 2003

[18] A J W Zendman D J Ruiter and G N P Van MuijenldquoCancertestis-associated genes identification expression pro-file and putative functionrdquo Journal of Cellular Physiology vol194 no 3 pp 272ndash288 2003

[19] A J G Simpson O L Caballero A Jungbluth Y T Chen andL J Old ldquoCancertestis antigens gametogenesis and cancerrdquoNature Reviews Cancer vol 5 no 8 pp 615ndash625 2005

[20] Y T Chen C Iseli C A Yenditti L J Old A J G Simpsonand C V Jongeneel ldquoIdentification of a new cancertestis genefamily CT47 among expressed multicopy genes on the humanX chromosomerdquoGenes Chromosomes and Cancer vol 45 no 4pp 392ndash400 2006

[21] O Hofmann O L Caballero B J Stevenson et al ldquoGenome-wide analysis of cancertestis gene expressionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 51 pp 20422ndash20427 2008

[22] P Van Der Bruggen C Traversari P Chomez et al ldquoA geneencoding an antigen recognized by cytolytic T lymphocytes ona human melanomardquo Science vol 254 no 5038 pp 1643ndash16471991

[23] U Sahin O Tereci H Schmitt et al ldquoHuman neoplasms elicitmultiple specific immune responses in the autologous hostrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11810ndash11813 1995

[24] M J ScanlanCMGordon BWilliamson et al ldquoIdentificationof cancertestis genes by database mining and mRNA expres-sion analysisrdquo International Journal of Cancer vol 98 no 4 pp485ndash492 2002

[25] M J Scanlan A J Simpson and L J Old ldquoThe cancertestisgenes review standardization and commentaryrdquo Cancer Im-munity vol 4 article 1 2004

[26] Y T Chen M J Scanlan C A Venditti et al ldquoIdentificationof cancertestis-antigen genes by massively parallel signaturesequencingrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 102 no 22 pp 7940ndash79452005

[27] Y H Cheng E W P Wong and C Y Cheng ldquoCancertestis(CT) antigens carcinogenesis and spermatogenesisrdquo Spermato-genesis vol 1 pp 209ndash220 2011

[28] P Chomez O De Backer M Bertrand E De Plaen T Boonand S Lucas ldquoAn overview of the MAGE gene family withthe identification of all human members of the familyrdquo CancerResearch vol 61 no 14 pp 5544ndash5551 2001

[29] Y Katsura and Y Satta ldquoEvolutionary history of the cancer Im-munity antigen MAGE gene familyrdquo PLoS ONE vol 6 no 6Article ID e20365 2011

[30] M T Ross D V Grafham A J Coffey et al ldquoThe DNAsequence of the human X chromosomerdquo Nature vol 434 pp325ndash337 2005

[31] E Fratta S Coral A Covre et al ldquoThe biology of cancertestis antigens putative function regulation and therapeuticpotentialrdquoMolecular Oncology vol 5 no 2 pp 164ndash182 2011

[32] O L Caballero and Y T Chen ldquoCancertestis (CT) antigenspotential targets for immunotherapyrdquo Cancer Science vol 100no 11 pp 2014ndash2021 2009

[33] U Sahin O Tureci Y T Chen et al ldquoExpression of multiplecancertestis antigens in breast cancer and melanoma basisfor polyvalent CT vaccine strategiesrdquo International Journal ofCancer vol 78 pp 387ndash389 1998

[34] S N Akers K Odunsi and A R Karpf ldquoRegulation of cancergermline antigen gene expression implications for cancerimmunotherapyrdquo Future Oncology vol 6 no 5 pp 717ndash7322010

[35] N Kouprina MMullokandov I B Rogozin et al ldquoThe SPANXgene family of cancertestis-specific antigens rapid evolutionand amplification in African great apes and hominidsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 9 pp 3077ndash3082 2004

[36] H Skaletsky T Kuroda-Kawaguchl P J Minx et al ldquoThemale-specific region of the human Y chromosome is a mosaic ofdiscrete sequence classesrdquo Nature vol 423 no 6942 pp 825ndash837 2003

[37] P E Warburton J Giordano F Cheung Y Gelfand and GBenson ldquoInverted repeat structure of the humangenome theX-chromosome contains a preponderance of large highly homol-ogous inverted repeated that contain testes genesrdquo GenomeResearch A vol 14 no 10 pp 1861ndash1869 2004

[38] IHGSC ldquoInternational human genome sequencing consor-tiumrdquo Nature vol 431 pp 931ndash945 2004

[39] Z Birtle L Goodstadt and C Ponting ldquoDuplication andpositive selection among hominin-specific PRAME genesrdquoBMC Genomics vol 6 article 120 2005

[40] T C Chang Y Yang H Yasue A K Bharti E F Retzel andWS Liu ldquoThe expansion of the PRAME gene family in EutheriardquoPLoS ONE vol 6 no 2 Article ID e16867 2011

[41] B J Stevenson C Iseli S Panji et al ldquoRapid evolution ofcancertestis genes on the X chromosomerdquo BMCGenomics vol8 article 129 2007

[42] N Kouprina V N Noskov A Pavlicek et al ldquoEvolutionarydiversification of SPANX-N sperm protein gene structure andexpressionrdquo PLoS ONE vol 2 no 4 article e359 2007

International Journal of Genomics 7

[43] Y Liu Q Zhu and N Zhu ldquoRecent duplication and positiveselection of the GAGE gene familyrdquo Genetica vol 133 no 1 pp31ndash35 2008

[44] M F Gjerstorff andH J Ditzel ldquoAn overview of the GAGE can-certestis antigen family with the inclusion of newly identifiedmembersrdquo Tissue Antigens vol 71 no 3 pp 187ndash192 2008

[45] A J W Zendman A A Van Kraats U H Weidle D J Ruiterand G N P Van Muijen ldquoThe XAGE family of cancertestis-associated genes alignment and expression profile in normaltissues melanoma lesions and Ewingrsquos sarcomardquo InternationalJournal of Cancer vol 99 no 3 pp 361ndash369 2002

[46] S Sato Y Noguchi N Ohara et al ldquoIdentification of XAGE-1 isoforms predominant expression of XAGE-1b in testis andtumorsrdquo Cancer Immunity vol 7 article 5 2007

[47] Y T Chen M Hsu P Lee et al ldquoCancertestis antigen CT45analysis of mRNA and protein expression in human cancerrdquoInternational Journal of Cancer vol 124 no 12 pp 2893ndash28982009

[48] Y T Chen A Chadburn P Lee et al ldquoExpression of cancertestis antigen CT45 in classical Hodgkin lymphoma and otherB-cell lymphomasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 7 pp 3093ndash3098 2010

[49] H J Heidebrecht A Claviez M L Kruse et al ldquoCharacteriza-tion and expression of CT45 in Hodgkinrsquos lymphomardquo ClinicalCancer Research vol 12 pp 4804ndash4811 2006

[50] B T Lahn and D C Page ldquoFour evolutionary strata on thehumanX chromosomerdquo Science vol 286 no 5441 pp 964ndash9671999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 International Journal of Genomics

[8] H Kaessmann N Vinckenbosch and M Long ldquoRNA-based gene duplication mechanistic and evolutionary insightsrdquoNature Reviews Genetics vol 10 no 1 pp 19ndash31 2009

[9] H Kaessmann ldquoOrigins evolution and phenotypic impact ofnew genesrdquo Genome Research vol 20 no 10 pp 1313ndash13262010

[10] A P Kozlov ldquoThe possible evolutionary role of tumors in theorigin of new cell typesrdquo Medical Hypotheses vol 74 no 1 pp177ndash185 2010

[11] J J Emerson H Kaessmann E Betran and M Long ldquoExten-sive gene traffic on the mammalian X chromosomerdquo Sciencevol 303 no 5657 pp 537ndash540 2004

[12] EW Sayers T Barrett D A Benson et al ldquoDatabase resourcesof the National Center for Biotechnology Informationrdquo NucleicAcids Research vol 40 pp D13ndashD25 2012

[13] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2011 httpwwwR-projectorg

[14] H SheffeThe Analysis of Variance Wiley New York NY USA1959

[15] H Sheffe ldquoMultiple testing versus multiple estimationImproper confidence sets Estimation of directions and ratiosrdquoThe Annals of Mathematical Statistics vol 41 no 1 pp 1ndash291970

[16] L A Goodman ldquoSimultaneous confidence intervals for con-trasts among multinomial populationsrdquo The Annals of Mathe-matical Statistics vol 35 pp 716ndash725 1964

[17] A J W Zendman J Zschocke A A Van Kraats et alldquoThe human SPANX multigene family genomic organizationalignment and expression in male germ cells and tumor celllinesrdquo Gene vol 309 no 2 pp 125ndash133 2003

[18] A J W Zendman D J Ruiter and G N P Van MuijenldquoCancertestis-associated genes identification expression pro-file and putative functionrdquo Journal of Cellular Physiology vol194 no 3 pp 272ndash288 2003

[19] A J G Simpson O L Caballero A Jungbluth Y T Chen andL J Old ldquoCancertestis antigens gametogenesis and cancerrdquoNature Reviews Cancer vol 5 no 8 pp 615ndash625 2005

[20] Y T Chen C Iseli C A Yenditti L J Old A J G Simpsonand C V Jongeneel ldquoIdentification of a new cancertestis genefamily CT47 among expressed multicopy genes on the humanX chromosomerdquoGenes Chromosomes and Cancer vol 45 no 4pp 392ndash400 2006

[21] O Hofmann O L Caballero B J Stevenson et al ldquoGenome-wide analysis of cancertestis gene expressionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 51 pp 20422ndash20427 2008

[22] P Van Der Bruggen C Traversari P Chomez et al ldquoA geneencoding an antigen recognized by cytolytic T lymphocytes ona human melanomardquo Science vol 254 no 5038 pp 1643ndash16471991

[23] U Sahin O Tereci H Schmitt et al ldquoHuman neoplasms elicitmultiple specific immune responses in the autologous hostrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 25 pp 11810ndash11813 1995

[24] M J ScanlanCMGordon BWilliamson et al ldquoIdentificationof cancertestis genes by database mining and mRNA expres-sion analysisrdquo International Journal of Cancer vol 98 no 4 pp485ndash492 2002

[25] M J Scanlan A J Simpson and L J Old ldquoThe cancertestisgenes review standardization and commentaryrdquo Cancer Im-munity vol 4 article 1 2004

[26] Y T Chen M J Scanlan C A Venditti et al ldquoIdentificationof cancertestis-antigen genes by massively parallel signaturesequencingrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 102 no 22 pp 7940ndash79452005

[27] Y H Cheng E W P Wong and C Y Cheng ldquoCancertestis(CT) antigens carcinogenesis and spermatogenesisrdquo Spermato-genesis vol 1 pp 209ndash220 2011

[28] P Chomez O De Backer M Bertrand E De Plaen T Boonand S Lucas ldquoAn overview of the MAGE gene family withthe identification of all human members of the familyrdquo CancerResearch vol 61 no 14 pp 5544ndash5551 2001

[29] Y Katsura and Y Satta ldquoEvolutionary history of the cancer Im-munity antigen MAGE gene familyrdquo PLoS ONE vol 6 no 6Article ID e20365 2011

[30] M T Ross D V Grafham A J Coffey et al ldquoThe DNAsequence of the human X chromosomerdquo Nature vol 434 pp325ndash337 2005

[31] E Fratta S Coral A Covre et al ldquoThe biology of cancertestis antigens putative function regulation and therapeuticpotentialrdquoMolecular Oncology vol 5 no 2 pp 164ndash182 2011

[32] O L Caballero and Y T Chen ldquoCancertestis (CT) antigenspotential targets for immunotherapyrdquo Cancer Science vol 100no 11 pp 2014ndash2021 2009

[33] U Sahin O Tureci Y T Chen et al ldquoExpression of multiplecancertestis antigens in breast cancer and melanoma basisfor polyvalent CT vaccine strategiesrdquo International Journal ofCancer vol 78 pp 387ndash389 1998

[34] S N Akers K Odunsi and A R Karpf ldquoRegulation of cancergermline antigen gene expression implications for cancerimmunotherapyrdquo Future Oncology vol 6 no 5 pp 717ndash7322010

[35] N Kouprina MMullokandov I B Rogozin et al ldquoThe SPANXgene family of cancertestis-specific antigens rapid evolutionand amplification in African great apes and hominidsrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 101 no 9 pp 3077ndash3082 2004

[36] H Skaletsky T Kuroda-Kawaguchl P J Minx et al ldquoThemale-specific region of the human Y chromosome is a mosaic ofdiscrete sequence classesrdquo Nature vol 423 no 6942 pp 825ndash837 2003

[37] P E Warburton J Giordano F Cheung Y Gelfand and GBenson ldquoInverted repeat structure of the humangenome theX-chromosome contains a preponderance of large highly homol-ogous inverted repeated that contain testes genesrdquo GenomeResearch A vol 14 no 10 pp 1861ndash1869 2004

[38] IHGSC ldquoInternational human genome sequencing consor-tiumrdquo Nature vol 431 pp 931ndash945 2004

[39] Z Birtle L Goodstadt and C Ponting ldquoDuplication andpositive selection among hominin-specific PRAME genesrdquoBMC Genomics vol 6 article 120 2005

[40] T C Chang Y Yang H Yasue A K Bharti E F Retzel andWS Liu ldquoThe expansion of the PRAME gene family in EutheriardquoPLoS ONE vol 6 no 2 Article ID e16867 2011

[41] B J Stevenson C Iseli S Panji et al ldquoRapid evolution ofcancertestis genes on the X chromosomerdquo BMCGenomics vol8 article 129 2007

[42] N Kouprina V N Noskov A Pavlicek et al ldquoEvolutionarydiversification of SPANX-N sperm protein gene structure andexpressionrdquo PLoS ONE vol 2 no 4 article e359 2007

International Journal of Genomics 7

[43] Y Liu Q Zhu and N Zhu ldquoRecent duplication and positiveselection of the GAGE gene familyrdquo Genetica vol 133 no 1 pp31ndash35 2008

[44] M F Gjerstorff andH J Ditzel ldquoAn overview of the GAGE can-certestis antigen family with the inclusion of newly identifiedmembersrdquo Tissue Antigens vol 71 no 3 pp 187ndash192 2008

[45] A J W Zendman A A Van Kraats U H Weidle D J Ruiterand G N P Van Muijen ldquoThe XAGE family of cancertestis-associated genes alignment and expression profile in normaltissues melanoma lesions and Ewingrsquos sarcomardquo InternationalJournal of Cancer vol 99 no 3 pp 361ndash369 2002

[46] S Sato Y Noguchi N Ohara et al ldquoIdentification of XAGE-1 isoforms predominant expression of XAGE-1b in testis andtumorsrdquo Cancer Immunity vol 7 article 5 2007

[47] Y T Chen M Hsu P Lee et al ldquoCancertestis antigen CT45analysis of mRNA and protein expression in human cancerrdquoInternational Journal of Cancer vol 124 no 12 pp 2893ndash28982009

[48] Y T Chen A Chadburn P Lee et al ldquoExpression of cancertestis antigen CT45 in classical Hodgkin lymphoma and otherB-cell lymphomasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 7 pp 3093ndash3098 2010

[49] H J Heidebrecht A Claviez M L Kruse et al ldquoCharacteriza-tion and expression of CT45 in Hodgkinrsquos lymphomardquo ClinicalCancer Research vol 12 pp 4804ndash4811 2006

[50] B T Lahn and D C Page ldquoFour evolutionary strata on thehumanX chromosomerdquo Science vol 286 no 5441 pp 964ndash9671999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Genomics 7

[43] Y Liu Q Zhu and N Zhu ldquoRecent duplication and positiveselection of the GAGE gene familyrdquo Genetica vol 133 no 1 pp31ndash35 2008

[44] M F Gjerstorff andH J Ditzel ldquoAn overview of the GAGE can-certestis antigen family with the inclusion of newly identifiedmembersrdquo Tissue Antigens vol 71 no 3 pp 187ndash192 2008

[45] A J W Zendman A A Van Kraats U H Weidle D J Ruiterand G N P Van Muijen ldquoThe XAGE family of cancertestis-associated genes alignment and expression profile in normaltissues melanoma lesions and Ewingrsquos sarcomardquo InternationalJournal of Cancer vol 99 no 3 pp 361ndash369 2002

[46] S Sato Y Noguchi N Ohara et al ldquoIdentification of XAGE-1 isoforms predominant expression of XAGE-1b in testis andtumorsrdquo Cancer Immunity vol 7 article 5 2007

[47] Y T Chen M Hsu P Lee et al ldquoCancertestis antigen CT45analysis of mRNA and protein expression in human cancerrdquoInternational Journal of Cancer vol 124 no 12 pp 2893ndash28982009

[48] Y T Chen A Chadburn P Lee et al ldquoExpression of cancertestis antigen CT45 in classical Hodgkin lymphoma and otherB-cell lymphomasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 107 no 7 pp 3093ndash3098 2010

[49] H J Heidebrecht A Claviez M L Kruse et al ldquoCharacteriza-tion and expression of CT45 in Hodgkinrsquos lymphomardquo ClinicalCancer Research vol 12 pp 4804ndash4811 2006

[50] B T Lahn and D C Page ldquoFour evolutionary strata on thehumanX chromosomerdquo Science vol 286 no 5441 pp 964ndash9671999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology