Genetic evidence for the existence of interleukin-23 and for variation in the interleukin-12 and interleukin-12 receptor genes in the horse

Comparative Biochemistry and Physiology, Part D 1 (2006) 179 – 186 www.elsevier.com/locate/cbpd

Genetic evidence for the existence of interleukin-23 and for variation in the interleukin-12 and interleukin-12 receptor genes in the horse Petr Kralik, Jan Matiasovic, Petr Horin * Institute of Animal Genetics, Faculty of Veterinary Medicine, Palacke´ho 1/3, CZ-612 42 Brno, Czech Republic Received 20 May 2005; received in revised form 11 September 2005; accepted 14 September 2005 Available online 9 December 2005

Abstract Immune loci, characterized by features reflecting their role in defense reactions and consequently related to evolutionary mechanisms, including polymorphisms or association with disease are suitable candidates for comparative analysis. Interleukin-12 and related cytokines are key molecules regulating natural and specific immune responses. In this study, we analyzed four horse IL12-related genes: IL23p19, IL12Rb2, IL12p40, and IL12p35. Genomic nucleotide sequence of the horse IL23 p19 sub-unit encoding gene was determined. The horse IL23p19 gene consists of four exons; its total mRNA length is 1004 bp, with a coding region of 579 bp. The predicted amino acid sequence of the horse IL23p19 sub-unit showed 88.0% sequence identity with the human sequence. A partial genomic sequence highly homologous to human IL12Rb2 suggesting existence of this gene in the horse was retrieved. Single nucleotide polymorphisms (SNPs) were identified in all four genes analyzed. PCR-RFLP genotyping was developed for selected SNPs. Inter-breed differences in allele and genotype frequencies were observed in IL12p35 SNP 242. The results showed that horse IL12-related genes are comparable to their counterparts in other mammalian species in terms of their structure and their genetic variation. D 2005 Elsevier Inc. All rights reserved. Keywords: Comparative genomics; Horse; Interleukin-12 family; Single nucleotide polymorphism

1. Introduction In humans and most other mammals, up to 5% of the genes are involved in defense mechanisms (Trowsdale and Parham, 2004). The contribution of host genetic factors to infectious diseases is one of fundamental issues in our understanding of their pathogenesis (Segal and Hill, 2003). Even minor variations in specific immune response genes could have an important impact on downstream responses and disease pathogenesis (Lazarus et al., 2002). As a result of an evolutionary interaction between the pathogen and the host, a large proportion of genetic variation in host resistance/susceptibility to infection seems to be caused by pathogens and/or host counter-adaptations (Slev and Potts, 2002). The vertebrate genome shows non-uniform patterns of sequence conservation. Also the immunity-related gene repertoire is dynamic and for each species tailored to the * Corresponding author. Tel.: +420 541 562 292; fax: +420 549 248 841. E-mail address: [email protected] (P. Horin). 1744-117X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2005.09.002

niche that the species occupied (Trowsdale and Parham, 2004). Comparative DNA sequence analysis can identify conserved sequences reflecting both functional constraints and the neutral mutational events that altogether shaped this genomic region. Small genomic regions that may be under purifying selection for a functional role are more highly conserved across multiple species than are neutrally evolving sequences (Frazer et al., 2003). Immune loci, characterized by features reflecting their role in defense reactions and consequently related to evolutionary mechanisms, including polymorphisms, clustering, rapid evolution or association with disease are thus suitable candidates for such comparative analysis (Trowsdale and Parham, 2004). Over six thousand years, the domestic horse has differentiated into many often old and specific domesticated breeds and populations suitable for comparative analysis. Currently, the horse genomics focuses on developing resources for identifying genes and mutations associated with different traits of significance, like inherited disease, mechanisms of disease, fertility, morphology, and perfor-

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mance traits. Besides the synteny map, the genetic linkage map, the cytogenetic map and the radiation hybrid map, the comparative map has become the fifth pillar of the future integrative horse map (Chowdhary and Bailey, 2003). Comparative genomics may serve as a powerful tool for identifying chromosome segments and specific genes involved in traits important for survival. Genes involved in resistance and/or susceptibility to infectious and other diseases thus are of great interest in terms of improvement and/or conservation breeding programs, as well as of other measures of disease control, like vaccine and drug design in horses. Interleukin-12 (IL12) is one of key molecules regulating immune reactions in vertebrates. The central role of IL12 in promoting Th1 response is now well established (Gately et al., 1998). IL12 induces interferon-gamma production by NK and T-cells. It also promotes the differentiation of naive CD4+ T cells into IFN-g producing T helper 1 (Th1) cells. IL12 thus regulates the development of adaptive immune cells and plays a central role in coordinating innate and adaptive immunity. Recently, new members of the IL12 family with novel important functions in the regulation of immune responses were discovered (reviewed by Brombacher et al., 2003; Trinchieri, 2003). The IL12 molecule is composed of two subunits, p40 and p35. Its receptor on the surface of Th lymphocytes (IL12R) is also formed with two subunits, designated IL12Rh1 and IL12Rh2 (Presky et al., 1996). IL12 p40 was found to associate not only with IL12 p35, but also with another molecule, p19, to form a new heterodimeric cytokine known as IL23 (Oppmann et al., 2000). IL23 binds to a receptor that is formed by IL12Rh1 and a new second chain, IL23R (Parham et al., 2002). IL23p19 has a homology to the IL12p35 (Oppmann et al., 2000). IL23 induces the same signaling pathways as IL12;

however, the activities of both molecules are distinct. Specific genes encode the IL12-related molecules and their receptors. Genes encoding the p40 and p35 sub-units in the horse were cloned and characterized (Nicolson et al., 1999). Structural and functional studies available (e.g. Nicolson et al., 1999; Heinzerling et al., 2001; McMonagle et al., 2001) suggest that the horse interleukin-12 could have similar roles in immune responses like in humans and other mammals. This, however, needs to be confirmed. A single nucleotide polymorphism (SNP) within the IL12 p40 sub-unit encoding sequence reported in the GenBank (Y11129) was genotyped as a PCRRFLP marker in our previous work, and significant interbreed differences in allelic frequencies were found (Horin et al., 2004). Besides IL12 p35 and p40 encoding genes, no other genes and/or molecules of the IL12 and IL12 receptor family were reported in horses so far. The objective of this study was to identify the IL23 p19 encoding gene in the domestic horse genome and to search for single nucleotide polymorphisms in four genes of the IL12 family in the domestic horse: IL12p40, IL12p35, IL12Rb2 and IL23p19.

2. Materials and methods 2.1. Primers Primers derived from the corresponding human nucleotide sequences and subsequent direct sequencing were used prior to designing horse specific primers for identifying the horse orthologues of human IL23p19 and IL12b2 genes (Table 1). Horse (Equus caballus) specific primers amplifying selected gene regions were designed for the search for single nucleotide polymorphisms (SNPs) (Table 1).

Table 1 Primers and annealing temperatures used in analysis of different horse IL12-related genes Gene

Primers (forward/reverse)

Annealing temperature (-C)

Cycles

GenBank accession numbers

IL12p40 IL12p40 IL12p40* IL12p40* IL12p40 IL12p40 IL12p35* IL12p35* IL23p19 IL23p19 IL23p19* IL23p19* IL23p19 IL23p19 IL12Rb2* IL12Rb2 IL12Rb2 IL12Rb2* IL12Rb2 IL12Rb2

5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V 5V

54 54 66 66 60 60 60 60 56 56 56 56 60 60 55 55 55 55 55 55

30 30 35 35 30 30 30 30 30 30 30 30 30 30 40 40 40 40 40 40

Y11129 Y11129 Y11129 Y11129 AY686644 AY686644 Y11130 Y11130 NM_016584 NM_016584 NM_016584 NM_016584 AY704416 AY704416 NM_001559 NM_001559 NM_001559 NM_001559 AY686643 AY686643

tgtatgttgtagaattggattggta 3V ctctgctgcttttgacactg 3V ctacaccagcggcttcttcatc 3V cttgccctggacctgaatagag 3V gcaattgttcccaaccaatc 3V tctgcgtagtgctcagaagaaa 3V gccaggcaaaccctagaat 3V ctgcatcagcttgtcaatgg 3V aaacaacaggaagcagcttacaa 3V tcacagccatctccacactg 3V tctgcttgcaaaggatccac 3V aaatacaataaataatcctccccaaac 3V ctggaggtgaacaacagaagg 3V cccagcagcttctcgtaaaa 3V gctccagaacagcctca/gaaa 3V gtgaatgtggatggaagtgaag 3V aaatttaacctggcagaagcaat 3V gttactgggccgatatctgagt 3V tggctgtataaaatgggatgtt 3V accaaataaacgaccaccaa 3V

*Primers used for RT-PCR.

P. Kralik et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 179 – 186 Table 2 PCR-RFLP genotyping: parameters of digestion with restriction enzymes Restriction enzyme

Units

Temperature (-C)

AciI TaqI SmaI BsrDI

10 20 20 2

37 65 25 65

181

951 gggccccacc ctcatgacct aatcaccccc aaagatccca cctcctaata

1001 ggatcacatt gggggttagg atttcaacat aggaattttg tggggacaca

1051 accattcaga ccatggcagc atctaagaat ggcaaagaac tgggtgggtc

1101 ttcagtgaat ggagtaggaa gagggtatcc attttctttc acggctttct

1151 tctctcccca gCCCTGCTTG CAAAGGATCC ACCAGGGCCT GGTTTTTTAC

2.2. PCR

1201 GAGAAGCTGC TGGGCTCAGA CATTTTCACA GGGGAGCCTT CTCTACTCCC

The PCR reaction mixture varied according to the gene analyzed. In general, the total reaction volume was 12.5 AL with 1.25 AL 10 Hot Star Taq buffer, 0.2 mM dNTPs, 10 pmol primers, 0.5 U Taq polymerase (TopBio) and 100 ng DNA. The PCR protocol consisted usually of an initial

1251 CAATGGCCCT GTGGACCAGC TTCACGCCTC CCTCCTGGGC CTCAGGCAAC

1301 TCTTGCAGgt atgaactagg ggcctggagg atgggggctt gcagggatca

1351 gagacaggga ctaggggtga aggatctaga gtcctctctg attgtgtcct

1 AAACAACAGG AAGCAGCTTA CAAACTGGAG GTGAACAACA GAAGGAACCA 1401 gtgtctttgc agCCTGAGGG TCACCACTGG GAGACTGAGC AGATTCCAAG

51 AACCAGAGCT CACAGAACAG AATCAGGCTG AGAGCAAGGG GAAGTGGGCA

101 GAGATTCCAC AGGGACTGAC TGGTGCAAGG CACAGAGCCA GCCAGGTTTG

1451 CCCCAGTCCC AGCCAGCCGT GGCAGCGCCT CCTTCTCCGC CCCAAGATCC 1501 TTCGCAGCCT CCAGGCCTTT GTGGCTGTAG CTGCCCGGGT CTTTGCCCAT

151 AGAAGCAGGC AGCAAGATGC TGGGGAGCAG AGCTGTGTTG CTGCTGCTGC

1551 GGAGCAGCAA CCCTGACCCC TTAAAGCCAG CAGCTTTAAG GATGGCACCC

201 TGCTCCTGTG GCCCCGGACT GCTCAGGCCC GGGCTGTGCC TGGAGGCAGT

1601 ACATCTATGG CTCAGCAATG CTAAGATGAA TCTATCAGCC CAGGCACCTA

251 AGCCCTGCCT GGGCTCAGTG CCAGCAGCTC TCACAGAAGC TCTGTACGCT

1651 CGAGCCAACA AGTTAATTTG TCCATTAATT CTAATGGGAC TTGCATATGT

301 GGCCTGGAGT GCACATCCAC CAATGGGACA TGTGgtgagt ggcagcctct

1701 TGAAAAATTA CCAGTACTGA CTGATTTCTG ATGCTGACCT AGCAAAAGGT

351 gggaccaagc aggactctat ccaagccctc caaggctgca ctggaagact

1751 TGAGTATTTA TTAGATGGGA TGGGAAAATT TGGGATTATT TATCCTCATG

401 ttggccttgg tggaagctgg agggttgaag gccatcaggg agtgagagag

1801 GCAGCAGTTT GGGGAGGATT ATTTATTGTA TTT 1833

a 451 gacaggagag tggggttcct gggagtgtca tgggcttgag ggtccaggtg

501 gggttcaaaa gtatttatct tatttcttca ctcttcctac cccctgctcc

551 attccagGAT CTACCAAGAG AAGAGGGAGA TGCTGAGACT ACAAATGATG

601 TCCCCCATAT CCAGTGCGAG GATGGCTGTG ATCCTGAAGG ACTCAGGGAC 651 AACAGTCAGg tactaagatg aggctggaaa taggggctgg agatatagct

701 aggcaccatg gtaaattagt aaaagttgta tctgtccttg tccgtttggc

751 ctgccataga atggacagct tataaacaac agaaatttat ttctcacagt

801 tctagaagct ggaagtctga gatgagggtg ccagcgtgtt gggtgagggc

Fig. 1 (continued).

denaturation at 95 -C for 2 min followed by 30 or 35 cycles of 94 -C at 30 s, annealing temperature 30 s, extension at 72 -C for 1– 3 min followed by a final extension at 72 -C for 10 min, followed by cooling to 4 -C. A complete list of primer pairs used in this study and the corresponding annealing temperatures are in Table 1. The PCR products obtained were cloned by the TOPO XL Cloning Kit (Invitrogen, USA) and sequenced by an ABI PRISM 310 Automated Sequencer (Applied Biosystems). All the sequences reported were determined based on at least two independent PCRs. 2.3. Reverse-transcription PCR (RT-PCR)

851 cctcttccca gctgcagact tctcattgta tcctcaggtg tcagaagggg

901 ctagggagct ctgtggatct cttttataag agcacctatc tcattcacga

Fig. 1. Genomic nucleotide sequence of the horse IL23p19 gene, including its complete coding region sequence. Human sequence derived primers are underlined; initiation and termination codons are double underlined. Intronic sequences are in lower-case letters. The polymorphic (SNP) nucleotide position identified is shaded.

Expression of the genes analyzed was investigated by RT-PCR. Purified peripheral blood monocytes and lymphocytes were used for isolating total RNA, and specific primers designed for this purpose were used in the RT-PCR assay (Table 1). Peripheral blood monocytes and lymphocytes were isolated according to a slightly modified protocol of Raabe et al. (1998). Cells were lysed using

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TriReagent (Sigma) and RNA was purified by chloroform – isopropanol extraction. For the RT-PCR reaction, the QuantiTec SYBR Green RT-PCR Kit (Qiagen) with 10 pmol of primers and 250 ng RNA was used. The RT-PCR consisted of a reverse transcription phase (50 -C, 30 min), followed by an initial denaturation at 95 -C 15 min, 40 cycles 15 s at 95 -C, annealing at the given temperature (Table 1) for 30 s and extension at 72 -C for 60 s, followed by a final extension at 72 -C for 10 min and cooling to 4 -C. Standard RT-PCR controls were used during the whole experiment. Negative controls did not contain nucleic acids, a constitutively expressed (MHCDQB) gene was used as a positive control, and intronspanning primers were used for excluding genomic DNA amplification. In all cases, expected amplifications and PCR products of expected size were obtained. 2.4. Sequence analysis The Blast analysis (Altschul et al., 1997) was used for identification of regions with high sequence similarity. Clustal X Multiple alignment (Thompson et al., 1997) was used for alignment of the sequences compared. The exon– intron boundaries were predicted by aligning the equine sequence with the human genomic sequences encoding the p40 (NT_023133), p35 (NT_005612), p19 (NT_029419), and Rh2 (NT_032977) sub-units, respectively. Phylogenetic analysis was performed by the neighbor-joining, parsimony and maximum likelihood methods using Phylip package, version 3.6 (Felsenstein, 2004; http://evolution.genetics. washington.edu/phylip.html). The 5VUTR nucleotide sequence of IL23p19 was analyzed using the Alibaba 2.1 software (http://www.gene-regulation.com/pub/programs/ alibaba2/) for the presence of transcription factors. The p19 protein sequence was analyzed for presence of glycosylation a phosphorylation sites using NetOGlyc, NetNGlyc and NetPhos on-line tools (http://folk.uio.no/ jethrolh/protein.html) and for a methylation sites using CpG plot tool (http://www.ebi.ac.uk/emboss/cpgplot/). 2.5. Single nucleotide polymorphisms The analysis of gene polymorphisms was done by selective re-sequencing and/or by PCR-RFLP. For this Table 3 Exon – intron organization of the horse IL23p19 gene (AY704416)

5VUTR Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Exon 4 3VUTR

Nucleotides

Length (bp)

1 – 166 167 – 334 335 – 557 558 – 659 660 – 1161 1162 – 1308 1309 – 1412 1413 – 1574 1575 – 1833

166 168 223 102 502 147 104 162 259

Horse Human Guinea pig Rat Mouse

....|....|....|....|....|....|....|....|....|....| 5 15 25 35 45 MLGSRAVLLLLLLLWPRTAQARAVPGGSSPAWAQCQQLSQKLCTLAWSAH .......M.....P.--...G...........T................. ....T..M.....P.--...T...S.S.N.S.T................. ..DC..II..W..P.--AT.GL...RS...D........RN......... ..DC...IM.W..P.--VT.GL...RS...D........RN..M...N..

Horse Human Guinea pig Rat Mouse

....|....|....|....|....|....|....|....|....|....| 55 65 75 85 95 PPMGHVDLPREEGDAETTNDVPHIQCEDGCDPEGLRDNSQPCLQRIHQGL .LV..M..-.....E...........G.....Q.......F......... .SV...EP....A.E...DY....L.G.....Q..K....F.....Y... T.V.QM..L....EE..KS...R...G.....Q..K....F.....R... A.A..MN.L...E.E..K.N..R.........Q..K....F.....R...

Horse Human Guinea pig Rat Mouse

....|....|....|....|....|....|....|....|....|....| 105 115 125 135 145 VFYEKLLGSDIFTGEPSLLPNGPVDQLHASLLGLRQLLQPEGHHWETEQI I...................DS..A.........S............Q.. ...QN...........P.F.D...S.........S......V.Q..P-.. ...KH..D............DS......T.....S......D.....Q.M A..KH..D....K...A...DS.ME...T.....S......D.PR..Q.M

Horse Human Guinea pig Rat Mouse

....|....|....|....| ....|....|....|....|....|... 155 165 175 185 195 PSPSPSQPWQRLLLRPKILRSLQAFVAVAARVFAHGAATLTP-----..L............F........................S.-----..L..N.........I.....F...........G........-----.RL....Q...S...S.........L.I.............EPLVPTA ..L.S..Q...P...S.........L.I.............EPLVPTA

Fig. 2. Predicted amino-acid sequence of the horse IL23 p19 protein and its comparison with the human, mouse, guinea pig and rat sequences. Dots and dashes indicate identity and deletions, respectively.

purpose, 1 AL of a PCR product were digested with the corresponding restriction enzyme overnight (Table 2), run in 6% PAGE gel and silver stained. PCR-RFLP markers developed were used for a population survey. Two different groups of horses were used for this purpose: a group of Thoroughbreds born in a single stud in the same year and a group of autochtonous Old Kladruber horses. Both of them were characterized previously for other immunity-related markers (Horin et al., 2004). Differences in allelic and genotype frequencies were tested by a standard v 2-test.

3. Results 3.1. The genomic sequence of horse IL23p19 Genomic nucleotide sequence of the horse IL23p19 encoding gene including its complete coding region was determined (Fig. 1). Genomic IL23p19 sequence showed the characteristic structure of a mammalian IL23p19 gene with four exons separated by three introns. No TATA box was found in the 5VUTR sequenced. The entire horse sequence reported here showed 87.28% identity with the corresponding part of the human sequence, while similarity of the coding sequences was 88.8%. Comparisons with mouse and rat genomic sequences were impossible, as only mRNA data are available for these species. Homologies of their coding sequences are apparent from comparisons of deduced amino acid data (see below). 3.2. Exon– intron organization of the horse IL23p19 gene Direct cDNA sequencing confirmed the exon boundaries predicted from alignments with the human IL23p19

P. Kralik et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 179 – 186

Human Guinea Pig 0.040111

Mouse

0.091721 0.110660

0.141978

0.025095

183

assigned accession number AY686643. The coding sequence obtained showed 84.4% homology with its human counterpart 87.41% with cattle, 86.71% with pig, and 86.29% with the dog sequence.

0.051948

3.5. RT-PCR

Horse

0.080695

Rat Fig. 3. Phylogenetic relationships indicated by relative distances between mammalian IL23p19 coding sequences based on the parsimony analysis algorithm (http://evolution.genetics.washington.edu/phylip.html).

sequence. The horse IL23p19 gene (AY704416) consisted of 4 exons; its total mRNA length was 1004 bp, with a coding region of 579 bp (Table 3). Within the 5VUTR, no methylation sites were found and a binding site for the nuclear factor kappaB was identified at position 100– 109. 3.3. The predicted amino acid sequence of horse IL23p19 The predicted amino acid sequence of the horse IL23p19 sub-unit showed 88.0% sequence identity with the human sequence, 78.6% for guinea pig, 74.3% for rat, and 70.2% for the mouse amino acid sequence (Fig. 2). No N-linked glycosylation sites were found. Two O-linked glycosylation sites at threonine positions 189 and 191 were found. Three serine (positions 89, 152 and 156) and one threonine (position 68) phosphorylation sites were found within the coding region of p19. 3.4. Partial genomic sequence homologous to human IL12Rb2 A partial genomic sequence homologous to human IL12Rb2 was retrieved with the primers used (Table 1). The sequence was deposited to the GenBank and was

IL23p19, IL12p40, and IL12p35 were found to be transcribed in monocytes and/or in cultivated and stimulated macrophages, while IL12Rb2 mRNA was identified only in peripheral blood lymphocytes. Blast analysis of the IL23p19 and IL12Rb2 coding sequences obtained against the horse EST database available at http://www.fungen.org/ did not identify any significant hit. GenBank searching only revealed homologies with human and macaque IL12Rb2 mRNA sequences (BU953711, BG203914, and CO648828, respectively). 3.6. Phylogenetic relationships Only four IL23p19 complete coding sequences were currently available for phylogenetic analysis and for comparison with the horse sequence (human, guinea pig, mouse, and rat AY359083, AB058509, AF301619, and AY055379, respectively). Phylogenetic trees constructed by using the neighbor-joining, maximum likelihood and parsimony algorithms showed all very similar patterns. As it is apparent from the example of the parsimony tree, the mouse and rat genes were phylogenetically closer between them than to other mammalian species (Fig. 3). 3.7. SNPs in horse IL12-related genes Single nucleotide polymorphisms were identified in all the genes analyzed. Each SNP was identified based on at least two independent PCRs from at least two different horses, confirmed by bi-directional sequencing and reconfirmed by appropriate restriction patterns, if possible. Moreover, bacterial clones with the appropriate allelic

Table 4 SNPs identified in horse IL12-related genes Locus

SNP position

SNP

Restriction enzyme

GenBank accession number

IL12p40 IL12p40 IL12p35 IL12p35 IL12p35 IL23p19 IL12RB2 IL12RB2 IL12RB2 IL12RB2

1453, exon 5 (syn)* 759, intron 3 242, intron 3 416, intron 3 563, exon 5 (syn) 422, intron 1 859, intron 4 1061, exon 5 (syn) 1077, exon 5 (nonsyn) 2426, intron 5

A to G A to T G to A del T G to A G to A C to G T to C C to G T to C

AciI None TaqI NT SmaI NT AciI NT BsrDI NT

AY686644 AY686641 AY686642 AY686642 AY686642 AY704416 AY686643 AY686643 AY686643 AY686643

syn: synonymous substitution. nonsyn: non-synonymous substitution. NT: not tested. *Reported already in the GenBank, accession number Y11129, analyzed in Horin et al. (2004).

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Table 5 Polymorphisms distribution in two horse breeds Polymorphism

Breed

Genotypes observed

n

Genotype frequencies

Allele frequencies

IL12p35

Old Kladruber N = 85

Thoroughbred N = 52

IL12p35

Old Kladruber N = 84

SNP 563

Thoroughbred N = 52

IL12Rb2

Old Kladruber N = 84

SNP 1077

Thoroughbred N = 52

65 18 2 9 29 14 0 19 65 2 17 33 79 6 0 41 10 1

0.765 0.212 0.024 0.173 0.558 0.269 0.000 0.226 0.774 0.038 0.327 0.635 0.929 0.071 0.000 0.788 0.192 0.019

0.871

SNP 242* p < 0.001

aa ab bb aa ab bb aa ab bb aa ab bb aa ab bb aa ab bb

sequence were always obtained by sub-cloning DNA from heterozygous animals. Exonic non-synonymous, exonic synonymous and intronic substitutions and/or deletions were identified. For selected SNPs, a PCR-RFLP genotyping method was developed (Table 4). The population distribution of IL12-related SNPs in two horse breeds is shown in Table 5.

4. Discussion The results provided evidence for the existence of two so far unidentified genes in the horse genome. A genomic nucleotide sequence including complete coding sequence, information on the genomic organization of IL23p19 and a partial genomic sequence homologous to human IL12Rb2 were obtained. Along with the data on their expression in the appropriate cell populations corresponding to theoretical expectations, the results suggest the existence of a so far unknown functional horse gene, encoding the p19 sub-unit of the horse interleukin-23. The functional significance of the putative binding, glycosylation and phosphorylation sites identified within the gene remains to be confirmed experimentally. However, their existence suggests a potential functional impact on the production and post-translation modifications of the protein synthesized. The number of species available for the phylogenetic comparison was too small to allow any general conclusions on the evolution of mammalian IL23p19 sequences. Similarity and patterns of the trees constructed by different methods might result from the fact that sequences from two phylogenetically closely related rodent species were available for comparisons with a very small and a more heterogenous group of other mammals. Another sequence referred as ‘‘Canis familiaris similar to interleukin 23, alpha subunit p19 precursor (LOC481110) mRNA’’ was derived

0.129 0.452 0.548 0.113 0.887 0.202 0.798 0.965 0.035 0.885 0.115

from an annotated genomic sequence (NW_876250) using gene prediction method GNOMON supported by EST evidence, available at http://www.ncbi.nlm.nih.gov/entrez/ viewer.fcgi?val=XM_538231.2 was not used for this purpose, since it has only been derived in silico. Homologies of several horse IL12-encoding genes with those of model mammalian species suggest that they could have similar role in the horse immune responses. The fact that also the protein coding sequence obtained with human IL12Rb2 primers showed a high degree of sequence similarity over more than 400 bp with the IL12Rb2 gene of different species, along with its expression pattern in appropriate peripheral blood lymphocytes strongly suggests the existence of a functional IL12Rb2 horse orthologue. Moreover, all the genes analyzed here including IL12Rb2 map physically to the corresponding evolutionary conserved homologous chromosome regions as assessed by FISH (Musilova et al., in press). Sequencing of the whole IL12Rb2 gene was not the goal of this study aiming to comparative genomic analysis of the IL23p19 gene and to identification of SNPs in selected IL12-related genes potentially underlying variations observed in the IL12-related immunity pathways. Therefore in all other genes, partial sequences were analyzed for polymorphisms (selective re-sequencing). Search for SNPs by selective re-sequencing is based on targeting functionally important parts of the genes analyzed (Lazarus et al., 2002). Our results showed existence of genetic variation in all these functionally important genes. Mendelian inheritance in families was confirmed for all the markers reported here (data not shown). Non-synonymous SNPs are candidates for further investigations on their role in disease mechanisms due to their direct effect on the resulting protein product. 5VUTR SNPs may affect gene expression and they thus also may be directly involved in disease mechanisms. Synonymous exonic and intronic SNPs may serve as association

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markers linked to causative mutations and for mapping purposes. All but one SNPs identified here have to be considered as linked markers, since they are either intronic or synonymous exonic substitutions. The functional importance of the 1077 C to G non-synonymous substitution within exon 5 of IL12Rb2 remains to be established. This polymorphism shows, along with 5VUTR SNPs and/or nonsynonymous substitutions identified in the horse NRAMP1, TNFA, TLR4 and CD14 genes (Matiasovic et al., 2002a,b; Horin et al., 2004; Vychodilova-Krenkova et al., 2005) that important immunity-related genes in horses are polymorphic, containing SNPs with potential functional impact. Inter-breed differences observed previously in IL12p40 SNPs (Horin et al., 2004) and in the IL12p35 SNP 242 reported here show that these polymorphisms might be useful markers for genomic analysis of immunity-related genes. Altogether, the markers reported thus represent a genomic tool for molecular dissection of horse immune responses. In humans, polymorphisms of IL-12 and IL12 receptor encoding genes were shown to be associated with variations in immune responses and with diseases (Seegers et al., 2002; Morahan et al., 2002; Van de Vosse et al., 2003). The results presented here show that the horse IL12-related genes are comparable to their counterparts in other mammalian species in terms of their structure and of their genetic variation. Future studies thus will focus on comparative functional aspects of the variation observed.

Acknowledgements Technical assistance of Mrs. E. Hanta´kova´ and of Mrs. V. Nova´kova´ is acknowledged. The work was supported by the Grant Agency of the Czech Republic projects 523/02/1526 and 524/00/1275 and by the Ministry of Education of the Czech Republic, project MSM 161700001.

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