1471-2164-8-129 1471-2164 Research article <p>Rapid evolution of cancer/testis genes on the X chromosome</p> Stevenson J Brian brian.stevenson@licr.org Iseli Christian christian.iseli@licr.org Panji Sumir sumir@sanbi.ac.za Zahn-Zabal Monique monique.zahn@licr.org Hide Winston winhide@sanbi.ac.za Old J Lloyd lold@licr.org Simpson J Andrew asimpson@licr.org Jongeneel C Victor victor.jongeneel@licr.org

Ludwig Institute for Cancer Research and Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland

South African National Bioinformatics Institute, University of the Western Cape, Bellville, 7535, South Africa

Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA

 BMC Genomics 1471-2164 2007 8 1 129 http://www.biomedcentral.com/1471-2164/8/129 17521433 10.1186/1471-2164-8-129 21 2 2007 23 5 2007 23 5 2007 2007 Stevenson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Cancer/testis (CT) genes are normally expressed only in germ cells, but can be activated in the cancer state. This unusual property, together with the finding that many CT proteins elicit an antigenic response in cancer patients, has established a role for this class of genes as targets in immunotherapy regimes. Many families of CT genes have been identified in the human genome, but their biological function for the most part remains unclear. While it has been shown that some CT genes are under diversifying selection, this question has not been addressed before for the class as a whole.

Results

To shed more light on this interesting group of genes, we exploited the generation of a draft chimpanzee (Pan troglodytes) genomic sequence to examine CT genes in an organism that is closely related to human, and generated a high-quality, manually curated set of human:chimpanzee CT gene alignments. We find that the chimpanzee genome contains homologues to most of the human CT families, and that the genes are located on the same chromosome and at a similar copy number to those in human. Comparison of putative human:chimpanzee orthologues indicates that CT genes located on chromosome X are diverging faster and are undergoing stronger diversifying selection than those on the autosomes or than a set of control genes on either chromosome X or autosomes.

Conclusion

Given their high level of diversifying selection, we suggest that CT genes are primarily responsible for the observed rapid evolution of protein-coding genes on the X chromosome.

Background

Cancer/testis (CT) genes are a growing family of genes defined by a unique pattern of expression: amongst normal tissues, they are expressed only in cells of the germ line and in embryonic trophoblasts, but their gene products are also found in a significant number of malignant cancers 1. The first CT genes were discovered because of the immune responses that they elicit in some cancer patients, and can thus be classified as CT antigens 23; systematic exploration of publicly available gene expression profiles (as documented in EST libraries, SAGE and MPSS data, and microarray experiments) uncovered a significant number of additional CT genes 45, against most of which immune responses have not yet been documented. Nevertheless, all CT genes are in principle attractive targets for cancer immunotherapy, because the gonads are immunoprivileged organs and anti-CT immune responses will therefore target tumours specifically. Vaccination using peptides derived from the NY-ESO-1 (CTAG1B) and MAGEA1 CT genes has already been proven to bring clinical benefits to melanoma patients 67.

CT genes comprise more than 240 members from 70 families, and can be subdivided into two broad categories based on chromosomal localization. CT-X genes are located on the X chromosome, are mostly members of gene families organized into complex direct and inverted repeats, and are expressed primarily during the spermatogonial stage of spermatogenesis 8. Non-X CT genes are located on autosomes, are mostly single-copy genes, and are expressed primarily during the meiotic and reduction division stages of spermatogenesis 8. Careful annotation of the sequence of the human X chromosome has revealed that as many as 10% of all genes present on the chromosome are members of known CT families 9; further analysis of the expression patterns of genes of unknown function located in repeated regions could even increase this estimate 5. The biological functions of most CT-X genes have not been characterized in any detail. However, evidence is emerging that the best studied of these, the MAGE genes, can act as signal transducing transcriptional modulators. Moreover, MAGE genes appear to be able to mediate proliferative signals 101112 and a member of the GAGE family has been shown to repress apoptosis 13, thus directly contributing to the malignant phenotype when aberrantly expressed in cancer. Available data suggest that many CT genes are involved in the re-programming of the transcriptional machinery that occurs during the transition from mitotic to meiotic division during spermatogenesis. It has been suggested that a similar re-programming may be responsible for some of the phenotype of malignant cancer cells 814.

There is mounting evidence that the evolutionary history of the human X chromosome is significantly different from that of autosomes. It contains a disproportionate number of tandem and interspersed segmental duplications, both direct and inverted, containing genes with a testis-specific expression pattern including many CT-X genes 9. These duplications are unstable in the genome, and subject to copy number polymorphisms, both within the human population and between humans and chimpanzees 1516. While its overall DNA sequence has diverged significantly less than that of autosomes since speciation of hominoids from chimpanzees 17, a significant proportion of protein-coding genes located on the X chromosome are under higher diversifying (positive) selection than those on autosomes 18. Genes located on the X chromosome are also the most abundant source of functional retrogenes in the primate lineage, and constitute a reservoir of genetic material for the generation of new genes and functions in this lineage, again with a bias toward testis-specific functions 1920.

For all of these reasons, it is of interest to trace the evolutionary history of CT genes, and particularly of the CT-X subset, and to measure the selective pressures that act on them. Many of the human CT-X genes do not have easily identifiable orthologues in the mouse, rat or dog genomes, precluding such an analysis among Eutheria using currently available genome data. For example, it has been shown that the large MAGE family of CT-X genes has expanded independently in the primate and rodent lineages 21. The recent availability of a draft genome for the chimpanzee has made it feasible to study the evolution of the CT genes within the primate lineage. We show here that the CT genes in general and the CT-X genes in particular are under strong diversifying pressure and amongst the fastest-evolving genes in the human genome.

Results

Identification of CT gene families in chimpanzee

To date at least seventy CT gene families, many with multiple members, have been identified in human. We took the opportunity afforded by the publication of the initial sequence of the chimpanzee genome 18 to ask whether CT genes were conserved in man's closest evolutionary neighbour. To this end we assembled a list of human transcript sequences representing all CT gene families, and searched for homologous sequences in the human and chimpanzee genomes. We expected that given the relatively short time elapsed since human-chimpanzee divergence (~ 6 million years ago 17) the human sequences would be able to detect CT gene homologues in the chimpanzee genome. Moreover, since the majority of CT genes isolated thus far were detected and characterized using transcript information via cDNA cloning protocols, performing the same search in human allowed us to identify all CT genes present in the current assembly of the human genome. We implemented a two-stage approach in order to accurately define the structure of each CT gene locus. First, we used MegaBlast 22 to search for regions homologous to the CT transcript sequences. Then we applied the SIBsim4 cDNA to genome alignment program (an improved version of sim4 23) to these regions to establish a gene structure from a locus-specific spliced alignment (see Methods). As can be seen in Table 1, almost all human CT families are found in chimpanzee, and the chromosomal locations of the CT genes in chimpanzee correspond to those in human. In terms of copy number, the biggest family, PRAME, is well represented in chimpanzee (37 genes), as are MAGEA (9 genes) CTAGE (15 genes), XAGE (12 genes) and SSX (8 genes). The number of CT genes in each family is probably underestimated because of the relatively low sequence coverage in the current version of the chimpanzee genome assembly. This is especially true for the X chromosome, where the sequence coverage is only about 2-fold 18, and where most of the human multi-gene CT families are located. Nevertheless, the current data indicate that some chimpanzee CT families (FTHL17/CT38, TSPY/CT78 and PRAME) may contain more members than in human.

<p>Table 1</p>

Number and chromosomal location of CT genes in human and chimpanzee

CT Number

Family Name

Human Chromosome

Human Gene Number

Chimpanzee Chromosome

Chimpanzee Gene Number

CT1

MAGEA

X

13 (0)

X

9 (0)

CT2

BAGE

5, 7, 9, 18, 21

7 (0)

7, 9, 18

4 (0)

CT3

MAGEB

X

7 (1)

X

7 (1)

CT4

GAGE

X

16 (0)

X

3 (0)

CT5

SSX

X

14 (0)

X

8 (0)

CT6

CTAG

X

3 (0)

X

1 (0)

CT7

MAGEC

X

2 (0)

X

1 (0)

CT8

SYCP1

1

1 (0)

1

1 (0)

CT9

BRDT

1

1 (0)

1

1 (0)

CT10

MAGEE

X

2 (2)

X

1 (1)

CT11

SPANX

X

11 (0)

X

4 (0)

CT12

XAGE

X

14 (0)

X

12 (0)

CT13

DDX43

6

1 (0)

6

1 (0)

CT14

SAGE

X

1 (0)

X

1 (0)

CT15

ADAM2

4, 8

2 (0)

4, 8

2 (0)

CT16

PAGE

X

7 (0)

X

6 (0)

CT17

LIPI

21

2 (0)

-

0 (0)

CT21

CTAGE

2, 6, 7, 9, 10, 13, 14, 18

21 (12)

2B, 6, 7, 9, 10, 13, 14, 18

15 (6)

CT24

CSAG

X

4 (0)

X

2 (0)

CT25

DSCR8

21

2 (0)

-

0 (0)

CT26

DDX53

X

1 (1)

X

1 (1)

CT27

CTCFL

20

1 (0)

20

1 (0)

CT28

LUZP4

X

1 (0)

X

1 (0)

CT29

CASC5

15

1 (0)

15

1 (0)

CT30

TFDP3

13, 15, X

4 (3)

15, X

2 (2)

CT32

LDHC

11

1 (0)

11

1 (0)

CT33

MORC1

3

1 (0)

3

1 (0)

CT34

DKKL1

19, 20

2 (1)

19, 20

2 (1)

CT35

SPO11

20

1 (0)

20

1 (0)

CT36

CRISP2

6

1 (0)

6

1 (0)

CT37

FMR1NB

X

1 (0)

X

1 (0)

CT38

FTHL17

X

4 (4)

X

5 (5)

CT39

NXF2

X

2 (0)

X

1 (0)

CT41

TDRD

6, 10

2 (0)

6, 10

2 (0)

CT42

TEX15

8

1 (0)

8

1 (0)

CT43

FATE1

X

1 (0)

X

1 (0)

CT44

TPTE

13, 21, Y

4 (0)

13

1 (0)

CT45

CT45

X

6 (0)

X

4 (0)

CT46

HORMAD1

1, 6

2 (1)

1, 6

2 (1)

CT47

LOC255313

X

12 (0)

X

2 (0)

CT48

SLCO6A1

5

1 (0)

5

1 (0)

CT49

TAG

5

1 (0)

5

1 (0)

CT50

LEMD1

1

1 (0)

1

1 (0)

CT51

HSPB9

17

1 (1)

17

1 (1)

CT53

ZNF165

6

1 (0)

6

1 (0)

CT54

SPACA3

17

1 (0)

-

0 (0)

CT55

CXorf48

X

3 (0)

X

1 (0)

CT56

THEG

19

1 (0)

19

1 (0)

CT57

ACTL8

1

1 (0)

1

1 (0)

CT58

NALP4

19

1 (0)

19

1 (0)

CT59

COX6B2

19

1 (0)

19

1 (0)

CT60

BC047459

15

2 (0)

Un

1 (0)

CT61

CCDC33

15

1 (0)

15

1 (0)

CT62

BC048128

15

1 (0)

15

1 (0)

CT63

PASD1

X

1 (0)

X

1 (0)

CT65

TULP2

19

1 (0)

19

1 (0)

CT66

AA884595

7

1 (1)

7

1 (1)

CT68

MGC27016

4

1 (0)

4

1 (0)

CT69

BC040308

6

1 (0)

6

1 (0)

CT71

SPINLW1

20

1 (0)

20

1 (0)

CT72

TSSK6

19

1 (1)

-

0 (0)

CT73

ADAM29

4

1 (0)

4

1 (0)

CT74

CCDC36

3

1 (0)

3

1 (0)

CT75

BC033986

2

1 (0)

2B

1 (0)

CT76

SYCE1

10

1 (0)

10

1 (0)

CT77

CPXCR1

X

1 (0)

X

1 (1)

CT78

TSPY1

Y

14 (0)

Y

22 (0)

CT79

TSGA

2, 21

3 (0)

2A

1 (0)

CT81

ARMC3

10

1 (0)

10

1 (0)

CTNA

PRAME

1, 22

36 (0)

1, 22, Un

37 (0)

CT gene families are presented in numerical order according to proposed nomenclature [1]. The largest family, PRAME, has not yet been assigned official CT designation. Total gene number for each family was determined according to sequence identity and completeness (see Methods). Numbers in brackets denote the number of intronless gene copies, which in the case of multi-exon genes may indicate putative retrocopy genes.

In order to investigate more closely the relatedness of CT genes in these two species, we sought putative human and chimpanzee orthologues for as many CT genes as possible, based on nucleotide sequence identity to the cognate human transcript sequence. Ninety-eight orthologous CT pairs were defined in this way (see Methods and additional file 1). The average identity of the human and chimpanzee orthologues to the human transcript sequences was 99.6% and 97.8%, respectively. Since we were interested in the characteristics of CT genes as a group, we also defined a group of human-chimpanzee orthologous non-CT control genes from chromosome X, where most of the CT genes are located, and from autosomal chromosomes 18 and 19 (see Methods). The reasons for choosing a limited set of control genes were two-fold: first, this allowed us to generate manually curated alignments of the same quality as for the CT genes, and second, it provided test and control groups of similar sizes for statistical analysis. The average identity of the human and chimpanzee control orthologues to the human transcript sequences was 99.6% and 98.7%, respectively. The finding that the chimpanzee and human CT orthologues were on average less closely related than the control orthologues (97.8% versus 98.7%; p < 2.2e-16 by a chi-squared test) suggested a possible difference in the divergence rates between the CT group and the control group. We tested this by analysing the substitution rates between human and chimpanzee ORF sequences (see below). Given the high accuracy of the human genomic sequence, the finding that the average human identity was less than 100% for both CT genes and non-CT control genes presumably reflects polymorphisms and/or sequencing errors in the original transcript sequences.

<p>Additional File 1</p>

Homology data on the human:chimpanzee putative orthologues used in this study. Excel spreadsheet presenting homology data on the human:chimpanzee putative orthologues.

Click here for file

CT genes on chromosome X are evolving faster than those on other chromosomes

We estimated the divergence rates of the CT genes from pairwise sequence alignments of the human and chimpanzee orthologues using phylogenetic analysis (PAML package 24). Mutations in a protein-coding gene can either have no effect (synonymous changes) or alter the sequence of the encoded protein (non-synonymous changes). The rate of synonymous changes (dS) indicates the background mutation frequency, while the ratio of the non-synonymous to synonymous mutation rates (dN/dS) indicates the type of evolutionary pressure acting on the gene. A dN/dS ratio value less than 1 suggests negative or purifying selection, a ratio equal to 1 suggests neutral evolution, and a ratio greater than 1 suggests positive or diversifying selection 25. To test what type of evolutionary pressure might be acting on the CT genes, we aligned the ORFs in the human-chimpanzee orthologue pairs and used the codeml program from the PAML package 24 to estimate the dN/dS ratios. Again, for comparison purposes, the control genes were subjected to an identical procedure. Figure 1 shows the distribution of dN/dS ratios for the CT genes and controls by chromosomal location. In contrast to the control genes, which show the distribution of ratios expected if most genes are under purifying selection, CT genes located on chromosome X have an excess of ratios greater than one. At the level of individual genes, SSX1, PAGE2B, SSX4, MAGEB2, GAGE4 and CPXCR1 have rate ratios greater than 2, indicative of strong evolutionary selective pressure acting on the gene products (Table 2). CT genes located on chromosomes other than chromosome X (CT-nonX) have a distribution of ratios skewed towards lower values, suggesting that this subgroup is evolving slower than the CT-X genes. In contrast, the majority of control genes, irrespective of chromosomal location, have rate ratios less than 0.5, suggestive of purifying selection. In addition, the nonsynonymous substitution rates for CT genes which had no synonymous changes between human and chimpanzee was on average higher than for the controls (see additional file 2).

<p>Table 2</p>

Nucleotide substitution rates estimated from alignments of human and chimpanzee orthologous CT ORFs

Gene Name

Refseq

Chromosome

dN

dS

dN/dS

ACTL8

NM_030812

1

0.0012

0.0170

0.0700

BRDT

NM_207189

1

0.0066

0.0071

0.9216

HORMAD1

NM_032132

1

0.0068

0.0104

0.6485

LEMD1

NM_001001552

1

0.0044

0.0327

0.1342

PRAMEF1

NM_023013

1

0.0162

0.0288

0.5624

PRAMEF2

NM_023014

1

0.0304

0.0317

0.9573

PRAMEF3

NM_001013692

1

0.0223

0.0269

0.8278

PRAMEF4

NM_001009611

1

0.0284

0.0305

0.9314

PRAMEF5

NM_001013407

1

0.0353

0.0586

0.6025

PRAMEF6

NM_001010889

1

0.0142

0.0149

0.9479

PRAMEF8

NM_001012276

1

0.0141

0.0262

0.5383

PRAMEF10

NM_001039361

1

0.0184

0.0262

0.7029

PRAMEF16

NM_001045480

1

0.0253

0.0236

1.0734

SYCP1

NM_003176

1

0.0050

0.0123

0.4093

BX103208

BX103208

3

0.0000

0.0346

0.0009

CCDC36

NM_178173

3

0.0065

0.0118

0.5502

MORC1

NM_014429

3

0.0071

0.0112

0.6325

CCDC110

NM_152775

4

0.0081

0.0142

0.5694

MGC27016

NM_144979

4

0.0017

0.0166

0.0994

SLCO6A1

NM_173488

5

0.0083

0.0093

0.8940

TAG1

AY328030

5

0.0001

0.1321

0.0009

BC040308

BC040308

6

0.0381

0.0004

∞

CRISP2

NM_003296

6

0.0034

0.0078

0.4355

DDX43

NM_018665

6

0.0046

0.0084

0.5422

TDRD6

NM_001010870

6

0.0029

0.0077

0.3756

ZNF165

NM_003447

6

0.0028

0.0083

0.3332

AA884595

AA884595

7

0.0000

0.0000

0.4503

BAGE2

NM_182482

7

0.0000

0.0000

0.4741

ADAM2

NM_001464

8

0.0090

0.0102

0.8787

TEX15

NM_031271

8

0.0064

0.0103

0.6188

BAGE

NM_001187

9

0.0000

0.0441

0.0009

ARMC3

NM_173081

10

0.0049

0.0142

0.3479

SYCE1

NM_130784

10

0.0073

0.0105

0.6979

TDRD1

NM_198795

10

0.0035

0.0085

0.4101

LDHC

NM_002301

11

0.0000

0.0070

0.0009

TPTE

NM_199261

13

0.0118

0.0095

1.2398

CTAGE5

NM_203356

14

0.0029

0.0082

0.3578

BC048128

BC048128

15

0.0077

0.0143

0.5355

CASC5

NM_170589

15

0.0084

0.0116

0.7226

CCDC33

NM_182791

15

0.0093

0.0192

0.4835

Klkbl4

XM_375358

16

0.0051

0.0109

0.4713

HSPB9

NM_033194

17

0.0112

0.0184

0.6077

CTAGE1

NM_172241

18

0.0108

0.0204

0.5311

COX6B2

NM_144613

19

0.0047

0.0138

0.3413

DKKL1

NM_014419

19

0.0055

0.0060

0.9034

NALP4

NM_134444

19

0.0090

0.0180

0.4981

THEG

NM_016585

19

0.0100

0.0091

1.1002

TULP2

NM_003323

19

0.0059

0.0056

1.0501

CTCFL

NM_080618

20

0.0124

0.0169

0.7316

SPINLW1

NM_181502

20

0.0134

0.0262

0.5122

SPO11

NM_012444

20

0.0044

0.0119

0.3679

PRAME

NM_006115

22

0.0191

0.0162

1.1798

CPXCR1

NM_033048

X

0.0104

0.0047

2.2411

CSAG1

NM_153478

X

0.0622

0.0006

∞

CSAG2

NM_004909

X

0.0163

0.0266

0.6138

CT45-2

NM_152582

X

0.0207

0.0002

∞

DDX53

NM_182699

X

0.0159

0.0109

1.4567

FATE1

NM_033085

X

0.0025

0.0142

0.1755

FMR1NB

NM_152578

X

0.0374

0.0228

1.6405

FTHL17

NM_031894

X

0.0150

0.0002

∞

GAGE4

NM_001474

X

0.0273

0.0117

2.3392

GAGE8

NM_012196

X

0.0244

0.0320

0.7617

LUZP4

NM_016383

X

0.0129

0.0138

0.9364

MAGEA10

NM_001011543

X

0.0083

0.0058

1.4380

MAGEA11

NM_001011544

X

0.0050

0.0055

0.9233

MAGEA12

NM_005367

X

0.0057

0.0222

0.2586

MAGEA2

NM_175743

X

0.0133

0.0126

1.0583

MAGEA4

NM_002362

X

0.0129

0.0086

1.4989

MAGEA5

NM_021049

X

0.0119

0.0001

∞

MAGEA8

NM_005364

X

0.0045

0.0074

0.6156

MAGEA9

NM_005365

X

0.0131

0.0171

0.7667

MAGEB1

NM_002363

X

0.0085

0.0129

0.6585

MAGEB2

NM_002364

X

0.0189

0.0068

2.7789

MAGEB3

NM_002365

X

0.0124

0.0001

∞

MAGEB4

NM_002367

X

0.0070

0.0133

0.5249

MAGEB5

XM_293407

X

0.0098

0.0117

0.8398

MAGEB6

NM_173523

X

0.0229

0.0157

1.4654

NXF2

NM_017809

X

0.0111

0.0125

0.8884

PAGE1

NM_003785

X

0.0102

0.0001

∞

PAGE2B

NM_001015038

X

0.0379

0.0117

3.2472

PAGE3

NM_001017931

X

0.0092

0.0087

1.0551

PAGE4

NM_007003

X

0.0000

0.0000

0.4989

PAGE5

NM_130467

X

0.0124

0.0001

∞

SAGE1

NM_018666

X

0.0096

0.0083

1.1487

SPANX-N2

NM_001009615

X

0.0216

0.0265

0.8131

SPANX-N4

NM_001009613

X

0.0151

0.0207

0.7276

SPANX-N5

NM_001009616

X

0.0000

0.0000

0.3869

SPANXD

NM_032417

X

0.1423

0.1107

1.2849

SSX1

NM_005635

X

0.0211

0.0057

3.7126

SSX2

NM_003147

X

0.0456

0.0373

1.2216

SSX4

NM_005636

X

0.0180

0.0059

3.0628

SSX5

NM_021015

X

0.0681

0.0622

1.0946

SSX8

NM_174961

X

0.0182

0.0002

∞

SSX9

NM_174962

X

0.0248

0.0208

1.1926

XAGE1

NM_133431

X

0.0145

0.0079

1.8487

XAGE2

NM_130777

X

0.0079

0.0001

∞

XAGE3

NM_133179

X

0.0046

0.0179

0.2556

XAGE5

NM_130775

X

0.0085

0.0118

0.7165

TSPY1

NM_003308

Y

0.0158

0.0241

0.6575

Synonymous (dS) and nonsynonymous (dN) nucleotide substitution rates were estimated using codeml from PAML [24] as described in Methods. Genes are presented by chromosomal location. '∞' denotes cases in which the dN/dS ratio cannot be calculated because the number of synonymous substitutions between the human and chimp sequences is zero.

 <p>Figure 1</p>

Distribution of dN/dS ratios for CT genes and controls

Distribution of dN/dS ratios for CT genes and controls. The proportion of genes in each category with ratios in intervals A-I is shown. The categories are: CT-X, CT genes on chromosome X (N = 33); CT-nonX, CT genes not on chromosome X (N = 49); Control-X, control genes on chromosome X (N = 64); Control-nonX, control genes not on chromosome X (N = 71). The intervals are: 0 ≤ A ≤ 0.25; 0.25 < B ≤ 0.5; 0.5 < C ≤ 0.75; 0.75 < D ≤ 1.0; 1.0 < E ≤ 1.25; 1.25 < F ≤ 1.5; 1.5 < G ≤ 1.75; 1.75 < H ≤ 2; 2 < I ≤ 4. Genes which had no synonymous changes (dN/dS denoted '∞' in Table 2) were omitted from the analysis.

 <p>Additional File 2</p>

Phylogenetic analysis of CT and control gene ORFs using codeml. Excel spreadsheet presenting data additional to that displayed in Table 2.

Click here for file

The apparent difference between the dN/dS distributions for the CT genes and the controls was assessed for significance using a nonparametric Mann-Whitney test, which indicates whether the medians of the two populations are significantly different. The difference in dN/dS values between all CT genes and all controls is highly significant with a p-value of 1.128e-11 (Table 3). Moreover, the difference between CT genes and the controls is significant whether the CT genes are located on chromosome X (p = 4.686e-10) or not (p = 1.498e-05). The distribution of dN/dS values is also significantly different for CT genes on chromosome X compared to those elsewhere (p = 2.812e-05), suggesting that there is stronger selective pressure on CT genes located on chromosome X. In contrast, there is no significant difference in the distribution of dN/dS ratios between the control genes located on chromosome X or elsewhere (p = 0.4962). Previous work has shown that the protein-coding genes on the hominid X chromosome have a higher average dN/dS value than other chromosomes 18. Our results suggest that the CT genes contribute strongly to this difference, and thus to the rapid evolution of protein-coding genes on the X chromosome.

<p>Table 3</p>

Significance of the differences in the distributions of dN/dS ratios between CT and control ORFs

Comparison

p-value

All CTs vs. All controls

6.22e-12

CT-Xs vs. Control-Xs

2.31e-10

Non-X CTs vs. Non-X controls

1.50e-05

CT-Xs vs. Non-X CTs

1.62e-05

Controls on X vs. Non-X controls

0.50

The distributions of dN/dS ratios from groups of CT and control ORFs were compared with each other, and any difference assessed using the non-parametric Mann-Whitney rank sum test [43]. Ratios denoted by '∞' in Table 2 were omitted from this analysis. For comparison, differences in the distributions were also assessed for significance using a parametric Welch two sample t-test; see additional file 3.

Discussion

Several recent publications have taken advantage of the chimpanzee draft genome to identify genes that are under diversifying selection in the primate lineage (26 and references therein). Their conclusions were concordant, in that they identified the X chromosome as containing a high number of positively selected genes, they found that positively selected genes are predominantly testis-specific, and that their functions are linked to gametogenesis as well as sensory perception and immunity against invading pathogens. Because most of these studies were performed at the whole genome level, they tended to focus on genes for which orthologues could be easily identified and pairwise alignments of coding regions generated automatically. This may explain why they failed to identify CT genes as a dominant group of positively selected genes. A review of recently published literature confirms that only a limited number of CT genes have been recognised as undergoing positive selection (Table 4). Moreover, a large proportion were identified through investigation of individual CT gene families (SPANX 27 and PRAME 28). In the present study, we have focused on the comparison between human and chimpanzee CT genes, with an emphasis on generating high-quality manually curated data. This was made necessary by the fact that many CT genes are located within segmental duplications and hence have multiple paralogues, and that we tried to be exhaustive in our analysis of all known CT gene families. Because of the large number of gaps that remain in the current assembly of the chimpanzee genome and the relatively high stringency we imposed on the extent of the alignments, we have certainly underestimated the number of CT homologues present in the chimpanzee genome, and some of the human:chimpanzee pairs may not correspond to true orthologues. However, neither of these problems should significantly affect the main conclusions of our study.

<p>Table 4</p>

Reports of positive selection pressure on CT genes

CT_family

Gene name

Human RefSeq

Reference

Present work#

CT1

MAGEA4

NM_002362

I

Yes

CT1

MAGEA5

NM_021049

I

Yes

CT1

MAGEA10

NM_021048

I

Yes

CT2

BAGE2

NM_182482

I

CT3

MAGEB2

NM_002364

I

Yes

CT3

MAGEB3

NM_002365

I

Yes

CT5

SSX1

NM_005635

I, III

Yes

CT5

SSX8

NM_174961

I, III

Yes

CT7

MAGEC2

NM_016249

I

CT7

MAGEC3

NM_138702

I

CT11

SPANX-N2

NM_001009615

III

CT11

SPANX-N3

NM_001009609

III

CT11

SPANX-N4

NM_001009613

III

CT11

SPANX-N5

NM_001009616

III

CT11

SPANXA

NM_013453

III

CT11

SPANXB

NM_032461

III

CT11

SPANXC

NM_022661

III

CT14

SAGE1

NM_018666

I, II

Yes

CT16

PAGE1

NM_003785

I

Yes

CT37

FMR1NB

NM_152578

I

Yes

CT38

FTHL17

NM_031894

I

Yes

CT48

SLCO6A1

NM_173488

I

CT55

CXorf48

NM_017863

I

CT56

THEG

NM_016585

I

Yes

CT63

PASD1

NM_173493

I

CT65

TULP2

NM_003323

I

Yes

CT77

CPXCR1

NM_033048

I

Yes

CT80

PIWIL2

NM_018068

I

CTNA

PRAME

NM_006115

I

Yes

CTNA

PRAME

cluster on chromosome 1

IV

Yes

Positive selection pressure on CT genes, from analysis of human and chimpanzee sequences, reported in: I, as defined by dN/dS > 1 [18, 33]. II, as defined by likelihood ratio test with p-value < 0.05 [35]. III, as defined by dN/dS > 1 [27] IV, inferred from dN/dS > 1 and sites modelling on human alignments [28] # Confirmed 16 previously reported positively selected CT genes, plus an additional 18 positively selected CT genes (see Table 2).

Given the close evolutionary kinship between humans and chimpanzees it is not surprising that all known CT gene families are shared between the two species. On the other hand, homologues of many CT antigens have not been found outside the primate lineage so far, and the available genome data are still too sparse to track the appearance of CT gene families during mammalian evolution. Even though the data are still incomplete, it is clear that most CT gene families are undergoing copy number expansions in the primate lineage, presumably driven by non-allelic homologous recombination between segmental duplications. The best-studied CT family in this respect is SPANX, which is present as a single-copy gene in rodents and has duplicated and acquired new sub-families in the primate lineage, including at least one (SPANX-C) found to be specific to humans on the basis of its genomic position 27. SPANX genes have been shown to have copy number polymorphisms in the human population, potentially linked to susceptibility to prostate cancer, and to undergo very rapid evolution affecting both dN and dS 29. An elegant study of the PRAME cluster on human chromosome 1 28 revealed the recent expansion in the human lineage of these genes via two large segmental duplications, and subsequent smaller duplications that may be polymorphic in the human population. The large MAGE family of CT antigens, which also comprises genes that do not show a CT expression pattern, has expanded in both the primate and rodent lineages, but independently 21. Our data also show that many MAGE genes are under diversifying selection (Table 2).

By definition, CT genes are expressed in testis, and for those for which data exists expression has been shown to be restricted to cells involved in spermatogenesis. It is believed that many CT genes are also expressed during oogenesis, but data on this process are still very sparse 3031. There is abundant evidence in the literature that many genes expressed predominantly during gametogenesis, as well as those implicated in reproduction in general (e.g. those encoding proteins found in the seminal fluid or expressed predominantly in the prostate) are undergoing positive selection during evolution 323334. In this respect, CT genes seem to behave much like other reproductive genes.

However, the CT-X genes are a special case, in that diversifying selective pressure seems more intense on this class. It is probable that the evolutionary pressures driving changes in the encoded protein sequences and those driving the expansion of the CT-X gene families are similar. Strikingly, the X chromosome is enriched in intrachromosomal tandem segmental duplications relative to autosomes 9. Several hypotheses have been put forward to explain why a subset of genes located on the X chromosome is evolving faster than those on autosomes 343536. Our data do not shed new light on this subject. However, it is interesting to note that CT-X genes contribute very significantly to the high average positive selection observed in protein-encoding genes on this chromosome, against a genomic background that is much more highly conserved than on the autosomes 17. One may speculate that transcriptional controls on recently duplicated genes could be relaxed relative to the parental copies, thereby allowing re-expression in tumours and the partial replication in these tumours of the transcriptional changes accompanying gametogenesis.

Conclusions

Essentially all human CT families have homologues at the same chromosomal locations in the chimpanzee genome. The copy numbers in the multi-gene CT families may differ between the two species but until a high-quality assembly of the chimpanzee genome is available this cannot be assessed in a reliable way. On the average, CT genes are under stronger positive selection than a set of randomly selected control genes. CT-X genes as a group are evolving very rapidly, not only relative to control genes on the X chromosome or on autosomes, but also relative to autosomal CT genes.

Methods

CT genes and human/chimpanzee genomic sequences

Human Reference sequence (RefSeq 37), or GenBank (where no RefSeq was available) entries were obtained for transcripts representing all documented CT gene families in the CT Gene Database 38. Transcript sequences were also obtained for additional candidate CT genes described in recent publications, which have not yet been added to the CT Gene Database. In some cases, multiple alternatively spliced transcript sequences from the same gene were selected to maximize sequence representation of the locus. Although PRAME has not been designated a CT gene, due to its trace level of expression in some normal adult tissues other than testis, it does exhibit the other main characteristics of CT genes, i.e. strong expression in the testis and up-regulation in various tumours, and was included in the set of CT genes selected for this study. Non-CT control genes were randomly chosen from lists of genes having a RefSeq identifier on chromosomes X, 18 (low gene density) and 19 (high gene density), generated using BioMart 3940. Control genes were selected from locations distributed uniformly along the lengths of the chromosomes to average out site-specific differences in mutation rates. The human (Homo sapiens) genomic sequence used was NCBI Build Number 36 (version 1, release date 9 March 2006), obtained from the NCBI. The chimpanzee (Pan troglodytes) genomic sequence used was NCBI Build Number 2 (version 1, release date 4 October 2006), also obtained from the NCBI.

Identification of CT gene loci in human and chimpanzee

CT gene loci were identified in both human and chimpanzee based on sequence identity between the human transcript sequences and human or chimpanzee genomic sequences. We used MegaBlast 22 to identify genomic regions homologous to the RefSeq sequences and SIBsim4 41 (an improved version of sim4 23) to produce high quality spliced alignments at those sites, from which locus-specific transcript sequences were generated. A gene was considered complete if the alignment contained at least 80% of the cognate transcript length or 80% of the annotated open reading frame (ORF), and had at least 85% identity to the human transcript sequence. Putative orthologues were identified as the sequences in human and chimpanzee genomes having the highest identity (and satisfying the 80% length threshold) to the same human transcript sequence. In many cases the poor quality (gaps, incorrect assembly) of the published chimpanzee genome sequence prevented us from finding a chimpanzee orthologue to the human gene. High quality sequence alignments for putative human/chimpanzee orthologues were obtained for 98 of the initial list of 135 CT genes (73%) and 153 of the 180 control genes (85%) selected randomly from chromosomes 18, 19 and X.

Divergence of CT genes

The genome-based transcript sequences derived from human and chimpanzee for each putative orthologous pair were aligned using clustalw (version 1.81 42), with gap extension penalties set to zero to allow gaps in the alignment arising from sequences missing in the chimpanzee assembly. Both sequences in the alignment were then trimmed to the extent of the human ORF based on annotation in the RefSeq or GenBank entry. Each nucleotide alignment was manually curated and revised, if necessary, to reflect the corresponding protein alignment. ORFs containing stop codons were dropped from the analysis. Rates of synonymous (dS; also known as Ks) and non-synonymous (dN; also known as Ka) substitutions between aligned ORFs were estimated using the codeml programme from the PAML package 24 with the F3x4 codon frequency model (and runmode = -2 in the codeml control file). Note that incomplete codons in either the human or the chimpanzee sequence are ignored by codeml. The statistical significance of differences in the distributions between human-chimpanzee divergence rates (dN/dS) among CT genes and controls was assessed using a Mann-Whitney (Table 3) or Welch two sample t-test (additional file 3) in the R package 43.

<p>Additional File 3</p>

Significance of the differences in the distributions of dN/dS ratios between CT and control ORFs using a parametric t-test. Distribution of dN/dS ratios assessed by parametric t-test. The results are qualitatively similar to those presented in Table 3 and confirm that the distribution of dN/dS values is different between CT genes and controls.

Click here for file

Abbreviations

CT – cancer/testis

CT-X – CT genes on chromosome X

dN – nonsynonymous substitution rate

dS – synonymous substitution rate

NCBI – National Center for Biotechnology Information

ORF – open reading frame

PAML – phylogenetic analysis by maximum likelihood

Authors' contributions

BJS, CI, LJO, AJS and CVJ designed the experiments. BJS wrote the software pipeline to identify human and chimpanzee CT genes and to produce ORF alignments. SP, MZ and WH scanned the literature for citations of positive selection. BJS and CVJ wrote the manuscript, which was read and approved by all authors.

Acknowledgements

We thank members of SIB Lausanne for discussions, and in particular Asa Wirapati and Frédéric Schutz for advice on statistical analysis of the phylogenetic data. This work was supported by the Ludwig Institute for Cancer Research.

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