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Rheumatology Advance Access originally published online on March 23, 2007
Rheumatology 2007 46(6):927-930; doi:10.1093/rheumatology/kel449
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Association of polymorphisms across the tyrosine kinase gene, TYK2 in UK SLE families

D. S. C. Graham, M. Akil1 and T. J. Vyse

Imperial College, Rheumatology Section, Hammersmith Hospital, Du Cane Road, London W12 0NN and 1Sheffield Centre for Rheumatic Diseases, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, UK

Correspondence to: T Y Vyse, Imperial College, Rheumatology Section, Hammersmith Hospital, De Cane Road, London W12 0NN. E-mail: t.vyse{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. This is a family-based association study to investigate the genetic contribution of tyrosine kinase 2 (TYK2 ) to disease susceptibility in 380 UK systemic lupus erythematosus (SLE) families, consisting of parents and affected offspring.

Methods. Genotyping was performed using the Sequenom platform on DNA from affected individuals and their parents. Haplotypes were constructed using Haploview from the founders, and family-based association was conducted using GENEHUNTER-TDT and Family-Based Association Test.

Results. There are two associated haplotypes across TYK2, both carrying alleles with distorted inheritance. One SNP shows individual association to SLE. This is the under-transmitted rare A allele of TYK2 SNP 6 (P = 0.004), which tags the under-transmitted haplotype 2 (P = 0.055). A second SNP shows a trend for association. This is the A allele of TYK2 SNP 13, which is unique to the over-transmitted haplotype 1 (P = 0.014). We defined a 2.8 kb core association region in TYK2, between these two variants, which narrows down the 5.7 kb gap in the study by Sigurdsson et al. (Sigurdsson S, Nordmark G, Goring HH et al. Polymorphisms in the tyrosine kinase 2 and interferon regulatory factor 5 genes are associated with systemic lupus erythematosus. Am J Hum Genet 2005;76:528–37).

Conclusions. We have shown association to SLE from individual SNPs and haplotypes in TYK2. The strongest individual association, which is carried on the associated haplotype, is from TYK2 SNP 6. The variant is located close to an intron/exon boundary, suggesting a role for mis-splicing events in molecular pathogenesis. The associated haplotype also carries a missense mutation at TYK2. Therefore it is likely that the allelic contribution of TYK2 to SLE is complex, our data confirm previous findings and provide additional resolution regarding the causal polymorphisms in this gene.

KEY WORDS: Systemic lupus erythematosus (SLE), Tyrosine kinase 2 (TYK2 ), Family-based association study


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Systemic lupus erythematosus (SLE) (OMIM 152700 [OMIM] ) is a multi-system complex autoimmune disease characterized by the production of autoantibodies against a diverse range of nuclear and cell surface autoantigens. Over recent years, the genetic basis of SLE has been well established [1].

Tyrosine kinase 2 (TYK2) is a 27.9 kb gene containing 25 exons, situated on chromosome 19p13.2, which forms part of a linkage region identified by Lindqvist et al. [2]. TYK2 is part of the janus kinase that binds to the interferon (IFN)-{alpha} receptor, IFNAR, on the cell surface of IFN-producing cells. TYK2 is bound to IFNAR in its inactive state, and after IFN-{alpha} binds to the IFNAR, TYK2 is phosphorylated and thereby activated [3]. Active TYK2 then phosphorylates IFNAR to allow binding of STAT3 and STAT5 [4]. The production of type I IFN and the regulation of IFN-inducible genes may have crucial importance in the aetiology of SLE, since raised levels of IFN-{alpha} are a well-established phenotype in SLE patients. This increase appears to be correlated with both disease activity and severity [5].

A previous association study in samples from Scandinavia by Sigurdsson et al. [6] suggested that alleles in TYK2 made a genetic contribution to the aetiology of in SLE. In this work, we have undertaken a family-based association study in a UK population across TYK2 in order to gain further understanding of the genetic contribution of TYK2 to SLE.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Family collection
The laboratory possesses a large collection of SLE families of predominantly European Caucasian (EC) origin. The study cohort consisted of 380 UK parent–proband trios, of which 358 are EC and 22 of Indo-Pakistani origin. All probands conformed to the ACR criteria for SLE [7]. Ethical approval was obtained from Multi-Centre Research Ethics Committee (MREC).

Selection of markers across TYK2
Eleven markers from TYK2 were taken from the recent publication by Sigurdsson et al. [6]. An additional nine markers across TYK2 were selected from the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/), to ensure good marker density across the gene, at a density of one SNP per 1.4 kb.

Genotyping methodology
All genotyping was performed using MALDI-TOF mass spectrometry, and analysis of the raw genotype data carried out using the MassArray Typer v3.4 software (Sequenom, Hamburg, Germany). Details of the assay designs are available on request.

Statistical analysis
All sample genotype and phenotype data were managed by, and analysis files generated with, the BC/GENE and BC/CLIN software (Biocomputing Platforms Ltd, Finland). Markers were excluded from the analysis if they showed <90% genotyping frequency, the HWE P-value was <0.05 and >5% families in the study cohort showed sporadic Mendelian errors. Haplotype patterns were constructed using HAPLOVIEW [8], using the Gabriel et al. [9] algorithm. TDT analyses to compare the observed and expected transmissions of alleles from heterozygous parents to affected offspring were performed using GENEHUNTER v2.0 beta [10, 11] and Family-Based Association Test (FBAT) [12, 13]. Correction for multiple testing was conducted using 1000 permutations run through HAPLOVIEW v3.31. The P-value generated represents the number of times the observed data would be expected to occur in the data set analysed.

Endophenotype (meaning a clinical subset within SLE) TDT analysis was performed on families where the affected individual had renal disease. A separate analysis was carried out for those families where the probands produced anticardiolipin autoantibodies. Familial clustering has been shown in our families for the presence of these autoantibodies (manuscript in preparation). The genetic effect was compared by calculating the odds ratio of the transmitted to untransmitted ratio (T:U). Endophenotype analyses were limited to avoid excess loss of statistical power.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Selection of informative markers in TYK2
From an initial 20 markers selected across TYK2, as described in the materials and methods, 18 SNPs generated viable assays for the Sequenom platform, since TYK2 SNPs 2 and TYK2 SNP 11 did not generate viable assays. TYK2 SNP 8 (rs2304255) was previously studied by Sigurdsson et al. [6], although was not reported to be associated with SLE. However, the assay was excluded from analysis in our UK study due to the high number of Mendelian errors (17% families). In one assay, TYK2 SNP 16 was monomorphic and TYK2 SNP 18 was of low allele frequency (<1%). A further three assays, TYK2 SNPs 8, 9 and 19 were excluded on the basis of having <90% genotyping, HWE P-values >0.05, and/or >5% families showing sporadic Mendelian errors. Consequently 13 assays (Table 1) were used for the TDT analyses.


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  		TABLE 1. Analysis of single SNPs across TYK2 by GENEHUNTER-TDT and FBAT

 
Haplotype structures across TYK2
In the haplotypes constructed from TYK2, there is a single haplotype block stretching across all the markers typed, consisting of six haplotypes at frequencies >3%. The haplotypes across the gene, illustrated in Fig. 1B, have captured 94.2% of the total chromosomes. The haplotypes were built from 358 EC parent–proband trios and 22 IP families, since in the parental samples for the EC and IP sub-populations there was no significant difference in minor allele frequency (MAF) for any SNP or in the inferred haplotype frequencies.


Figure 1
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  		FIG. 1. Genomic organization, haplotype architecture and haplotype-TDT across the human TYK2 gene. (A) The genomic organization of the human TYK2 gene. The exons are marked with black boxes, with the 5' and 3' UTRs as white boxes. The polymorphisms selected for the study are numbered 1–20. (B) The haplotype block structure across TYK2, as constructed using Haploview, from 358 EC parent–proband trios and 22 Indo-Pakistani trios. There is a single haplotype block across the gene, consisting of six haplotypes, numbered 1–6 down the left-hand side. The haplotypic frequency is shown to the right of each haplotype. The markers numbers are shown above the haplotypes and correspond to the SNPs illustrated in Fig. 1A. The haplotype-tagging alleles are indicated by asterisks under the marker numbers. Haplotype TDT was performed using GH-TDT on the joint EC-IP UK samples. The common haplotype (1) is over-transmitted (P = 0.014). The over-transmitted A allele of SNP 13, which is outlined by a smaller white box, is unique to the over-transmitted haplotype 1. The core association region between TYK2 SNPs 6 and 13 is outlined by a solid box. There is under-transmission of haplotype 2 (P = 0.054), with the core association region being outlined by a discontinuous box. Haplotype 2 uniquely carries the under-transmitted A allele of SNP 6, also shown in a smaller white box on the haplotype. The SNPs included in both the UK SLE study and the HAPMAP projects are indicated by an inverted triangle under the haplotypes. (C) SNPs 1, 3, 5–7, 13 and 15 were typed as part of the Sigurdsson et al. [6] study and also the UK SLE study. The associated alleles from the UK SLE data and those from Sigurdsson et al. [6] are superimposed on the haplotype structure from the UK SLE study. As in Fig. 1B, the associated alleles in the UK data set are shown inside the white boxes. The over-transmitted alleles of SNPs 7 and 15 are illustrated inside the white circles.

 
TDT analysis for individual SNPs in TYK2
TDT analysis was performed in the 358 EC parent–proband trios and 22 IP families using GENEHUNTER-TDT (GH-TDT) and confirmed using FBAT. The results of the GH-TDT analysis for variants across TYK2 are presented in Table 1. There is association of two markers with SLE. These are an over-transmission of the A allele of TYK2 SNP 13 and an under-transmission of the rare A allele of TYK2 SNP 6 (Table 2). The P-values from GH-TDT were 0.004 (SNP 6) and 0.057 (SNP 13). To correct for multiple hypothesis testing, we generated empirical P-values for association. Using these criteria, a single marker, SNP 6, showed association (P = 0.05). TYK2 SNP 6 is situated 36 bp away from the 3' end of intron 7 and may modulate splicing.


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  		TABLE 2. Haplotype-TDT in TYK2

 
Haplotype-TDT analysis
Haplotype-TDT using GENEHUNTER was performed on the haplotypes constructed across TYK2 (Fig. 1B). The commonest haplotype, haplotype 1, is over-transmitted with a P-value of 0.01 (Table 2), in which the unique element appears to lie between SNPs 6 and 13. There is also under-transmission of haplotype 2, with a P-value of 0.055, which carries the under-transmitted unique A allele of SNP 6.

Endophenotype analysis across both TYK2
Endophenotype analysis was carried for each variant across TYK2 in families where the proband had renal disease (n = 129), or possessed either IgG or IgM anticardiolipin autoantibody (n = 130). No significant difference in the odds ratio was seen for any variant for families where the proband had renal disease, or possessed either anticardiolipin autoantibody (P > 0.18; results not shown in detail).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Haplotype structure in UK SLE families
At TYK2, in the SLE families, there is a strong spine of LD running across the gene, resulting in a single haplotype block, composed of six haplotypes, which is comparable to that observed in HAPMAP CEU families. TYK2 is located between cell division cycle 37 homologue (CDC37) and intracellular adhesion molecule 3 (ICAM3). CDC37 is a co-chaperone of tyrosine kinases such as JAK1, which is important in the IFN induction of gene expression [14] and ICAM3 is a lymphocyte adhesion molecule expressed on resting lymphocytes [15]. Using the HAPMAP haplotypes, it was seen that there were clear haplotypic break-points between TYK2 and both of these neighbouring genes, with r2-values of <0.003. The lack of a large number of low-frequency haplotypes at TYK2 and the high percentage of the two commonest haplotypes across the gene indicate that it is unlikely we have missed a major risk haplotype, given the high density of polymorphisms interrogated (<1 per 2kb).

Comparison of association across TYK2 in UK with previous reports
In the UK data, we have demonstrated that in the centre of TYK2 there is a core region of association stretching over 2.8 kb, from intron 7 to intron 11. This region is bounded by the variants showing the strongest association in the UK data, TYK2 SNP 6 in intron 7 (P = 0.004), and a second association with TYK2 SNP 13 at the boundary of exon 10 and intron 11 (P = 0.057). The two versions of this core association region differentiate the two major associated haplotypes of TYK2, so that an AAGAG sub-haplotype uniquely demarcates the under-transmitted haplotype 2 (P = 0.055), and the GCTAA allelic combination is uniquely carried by the over-transmitted haplotype 1 (P = 0.014). Within this core region of association, all the markers, with the exception of TYK2 SNP 12, delineate the associated haplotype. Although the associations in TYK2 are weak, it likely that the gene does make a contribution to the aetiology of SLE, since the permuted P value for the polymorphism showing the strongest association (TYK2 SNP 6) was 0.05, with 1000 permutations. Furthermore, TYK2 SNPs 6 is in strong LD with TYK2 SNP 8 (r2 = 0.91), which was the variant showing the strongest association in the study by Sigurdsson et al. [6]. In addition, our data are compatible with the association of SNP 7, the missense polymorphism in exon 8. Although we do not see an association with SNP 7 in isolation, it is present on a number of neutrally transmitted low-frequency haplotypes. However, it is unlikely that TYK2 SNP 15, which was significantly associated in the Scandinavian and Finnish samples, makes a major contribution to the genetic effect seen with TYK2 in the UK SLE families, since it lies outside the core association region and the alleles do not segregate with haplotypes 1 and 2 (Fig. 1C). Although we cannot fully exclude an effect from TYK2 SNP 15 due to limited power as a result of its low allele frequency.

The original report of association in TYK2 demonstrated that the association could lie within a 5.7 kb region between TYK2 SNPs 7 and 15. By increasing the marker density across the gene, we have narrowed this core region of association to a 2.8 kb interval between TYK2 SNPs 6 and 13, since for all other variants both sides of these SNPs, the alleles are identical on the major associated haplotypes. Although, we cannot exclude the possibility that additional polymorphisms outside this core region are in LD with polymorphisms within it. Functionally, the SNPs in TYK2 showing the best association in the UK population are in close proximity to intron/exon boundaries, with TYK2 SNP 6 being 36 bp away from the 3' end of intron 7 and TYK2 SNP 13 located 7 bp away from the start of intron 11. Comparison of the sequences around these two variants using the GenomeVISTA browser [16, 17], supports the prediction that these SNPs may play an important role in the function of the protein, since alignment of the human genome with those for mouse (May 2004, SLAGEN), rat (June 2003, SLAGEN) and dog (July 2004, SLAGEN), showed >50% sequence conservation. For the missense F/V variant TYK2 SNP 7, which showed significant association in a previous study [6], the valine residue was evolutionarily conserved, which corresponds to the over-transmitted common C allele. The high level of sequence conservation for all these associated polymorphisms may reflect the fact that the associated SNPs are either in exons or at intron/exon boundaries and are therefore predicted to play an important role in the function of the protein.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was funded by the Wellcome Trust, through a Senior Fellowship awarded to T.J.V. We acknowledge the work of Paul Spencer and Andrew Wong in recruiting patients and families into the study and we would like to thank our clinical colleagues for helping us recruit study participants. Our thanks and appreciation is extended to all the patients and their relatives for generously donating blood samples and all the general practitioners and practice nurses for collecting them: many thanks to Prof. John Whittaker for his advice concerning the statistical analysis and to Dr David Perkins for maintaining and customizing the computerized database.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 29 November 2006; revised version accepted 15 December 2006.
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