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The SH2D2A gene and susceptibility to multiple sclerosis
Journal of Neuroimmunology 197 (2008) 152 – 158 www.elsevier.com/locate/jneuroim
The SH2D2A gene and susceptibility to multiple sclerosis Åslaug R. Lorentzen a,c,⁎, Cathrine Smestad b , Benedicte A. Lie c , Annette B. Oturai d , Eva Åkesson e , Janna Saarela f , Kjell-Morten Myhr g,h , Frode Vartdal c,i , Elisabeth G. Celius b , Per S. Sørensen d , Jan Hillert e , Anne Spurkland j , Hanne F. Harbo b a
e
Department of Neurology, Faculty Division Ullevål University Hospital, University of Oslo, Oslo, Norway b Department of Neurology, Ullevål University Hospital, Oslo, Norway c Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway d Danish MS Research Center, Department of Neurology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Division of Neurology, Department of Clinical Neuroscience, Karolinska Institute at Karolinska University Hospital, Huddinge, Stockholm, Sweden f Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland g Department of Neurology, Haukeland University Hospital, Bergen, Norway h Department of Clinical Medicine, University of Bergen, Bergen, Norway i Faculty Division Rikshospitalet University Hospital, University of Oslo, Oslo, Norway j Institute of Basal Medical Science, University of Oslo, Oslo, Norway Received 10 January 2008; received in revised form 25 April 2008; accepted 29 April 2008
Abstract We previously reported an association between the SH2D2A gene encoding TSAd and multiple sclerosis (MS). Here a total of 2128 Nordic MS patients and 2004 controls were genotyped for the SH2D2A promoter GA repeat polymorphism and rs926103 encoding a serine to asparagine substitution at amino acid position 52 in TSAd. The GA16–rs926103⁎A haplotype was associated with MS in Norwegians (OR 1.4, P = 0.04). A similar trend was observed among Danes. In the independent Norwegian, Danish and Swedish sample sets the GA16 allele showed a combined OR of 1.13, P = 0.05. Thus, the present study shows that the SH2D2A gene may contribute to susceptibility to MS. © 2008 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; SH2D2A; Autoimmunity; Disease susceptibility; TSAd
1. Introduction Multiple sclerosis (MS) is presumed to be an autoimmune disease affecting the central nervous system. The disease leads to demyelination and axonal damage, and is one of the most common causes of neurological disability in young adults in western countries. Both environmental and genetic factors contribute to susceptibility and pathogenesis of the disease. Many potential susceptibility genes have been suggested, and until recently the human leukocyte antigen (HLA) class II gene
⁎ Corresponding author. Institute of Immunology, Rikshospitalet University Hospital, 0027 Oslo, Norway. Tel.: +47 23 07 35 00; fax: +47 23 07 35 10. E-mail address: [email protected] (Å.R. Lorentzen). 0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.04.037
region was the only region that was firmly established to be associated with MS (Jersild et al., 1973; Olerup and Hillert, 1991; Sawcer et al., 2005). However, the genetic susceptibility probably involves many different genes as postulated in the common allelic variants hypothesis (Dyment et al., 2004; Oksenberg and Barcellos, 2005). Recently, in a whole genome association study performed by typing 334 923 SNPs, an association with MS was observed for IL2RA and IL7RA in addition to genes in the HLA region (Hafler et al., 2007). We previously reported an association with MS to the SH2 domain protein 2A (SH2D2A) gene, which is located on chromosome 1q23.1. The gene encodes the T cell specific adaptor protein (TSAd), which is expressed in activated T cells (Spurkland et al., 1998; Sundvold et al., 2000), as well as in NK cells (Nejad et al., 2004) and endothelial cells (Matsumoto et al.,
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2005; Wu et al., 2000). Although the function of TSAd in T cells is not fully understood, TSAd is important for normal differentiation and activation of T cells. T cells from mice lacking the SH2D2A gene showed reduced capacity for proliferation and reduced production of the T helper cell type one (TH1) cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ) (Rajagopal et al., 1999). These mice spontaneously developed a lupus-like autoimmune disease (Drappa et al., 2003). The SH2D2A gene is located in a chromosomal region that has been implied in the genetic susceptibility to autoimmune diseases in mice (Johannesson et al., 2005). It is not yet known whether TSAd-knock-out mice are more prone to develop organ specific autoimmune diseases. However in the diabetes prone NOD mice, reduced SH2D2A expression may be linked to a defect in the induction of central tolerance (Zucchelli et al., 2005). Furthermore, a meta-analysis of published gene expression studies in MS patients revealed that T cells from MS patients display reduced expression of the SH2D2A gene (Fernald et al., 2005). A dinucleotide (GA) repeat polymorphism located in the SH2D2A promoter gene region, at position − 340 from the initiator ATG codon (Dai et al., 2000), has been found to be associated with susceptibility to MS in a Norwegian case– control cohort and in Nordic multiplex families (Dai et al., 2001). In Nordic MS sib pair families, the GA16 allele was overtransmitted, and among unrelated Norwegians the frequency of patients carrying homozygous GA13 and/or GA16 genotypes was higher than that observed in healthy controls. A similar association was observed in juvenile rheumatoid arthritis (Smerdel et al., 2004). Expression of TSAd in activated T cells was lower in healthy individuals carrying a homozygous SH2D2A GA13 and/or GA16 genotype (Dai et al., 2001), suggesting that the length variation of the promoter GA repeat could have a direct functional significance. Alternatively, the GA repeat polymorphism may be in linkage disequilibrium (LD) with some other disease promoting polymorphisms within the SH2D2A gene region. To further explore the contribution of the SH2D2A gene in MS, we fine mapped the SH2D2A gene in Norwegian MS cases and controls and extended our studies by including a large sample set of Nordic MS patients and controls. 2. Materials and methods 2.1. MS patients and controls In total, 2128 unrelated Nordic MS cases and 2004 healthy controls were included in the present study, from which DNA was extracted from blood samples using standard methods. This included 624 unrelated MS patients and 562 healthy controls from Norway. Some of these samples had previously been genotyped for the GA repeat polymorphism in the promoter region of the SH2D2A gene (MS n = 313 and control n = 277) (Dai et al., 2001). An additional 1027 newly collected Norwegian controls were genotyped for the SNP rs926103 only. All Norwegian MS patients were diagnosed as definite MS according to the Poser criteria (Poser et al., 1983) or the McDonald criteria (McDonald et al., 2001). The female:male
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ratio was 1.4:1, and the mean age at onset was 31.1 years (range 10–62 years). The initial disease course was relapsing remitting MS (RR-MS) in 82% of the patients and primary progressive MS (PP-MS) in 18% of the patients. The Norwegian control samples were all randomly collected among healthy blood donors recruited through the Norwegian Bone Marrow Donor Registry. The female:male ratio was 1.4:1 and 1.5:1 respectively in the two sets of controls. The remaining Nordic 1504 MS cases and 1442 controls were recruited from Denmark, Sweden and Finland. All the MS cases fulfilled either the definite MS criteria of Poser or the MS criteria of McDonald. In these MS samples the female:male ratio was 2.6:1, the mean age at onset was 31.8 years (range 11– 63 years), and 91.4% were classified initially as RR-MS. The control samples from the same three Nordic countries had a female:male ratio at 0.9:1. The regional Ethics Committees approved the protocol, and informed consent was obtained from each individual. 2.2. Marker selection and genotyping The GA repeat polymorphism in the promoter region of the SH2D2A gene was amplified from all available samples (Norwegian, Danish, Swedish and Finnish cases and controls) as previously described (Dai et al., 2000, 2001). The microsatellite alleles were identified by fragment length analysis on an ABI Prism® 377XL sequencer or ABI Prism® 3730 DNA sequencer (Applied Biosystems, Foster city, CA, USA). Data were analysed using GeneMapper® version 3.5 or 3.7 or Gene Scan® 2.1 and the Genotyper® 2.0 software. The success rates for genotyping of the GA repeat polymorphism were above 94.2% for all case–control cohorts. Single nucleotide polymorphisms (SNPs) were selected within a 17 566 base pair (bp) region, stretching from two kb upstream to five kb downstream of the SH2D2A coding region. In the target region between 37 and 63 SNPs were reported in the HapMap (HapMap Data Rel 20/phaseII Jan 06, on NCBI B35 assembly, dbSNP b 125) (http://www.hapmap.org/index.html), UCSC (Assembly May 2004) (http://genome.ucsc.edu/index. html), Entrez SNP (http://www.ncbi.nlm.nih.gov/) or Ensembl (http://www.ensembl.org/index.html) databases. Among these, 47 SNPs had allele frequencies or heterozygosity data available in the databases. By using the Haploview tagging tool (r2 N 0.8, minor allele frequency (MAF) N 0.1) five SNPs were selected to tag the SH2D2A region (Imported HapMap data in Haploview was used). None of the selected SNPs displayed a MAF below 0.10 in the database. When tagging the SH2D2A gene region for haplotypes, the r2 between the selected SNPs was estimated to be below 0.80. Two of the chosen SNPs were previously genotyped in a limited number of the Norwegian MS patients and healthy controls (rs2768764 in the SH2D2A promoter (− 446 in Dai et al., 2001) and rs1800600 in intron 2 (IVS2 + 21 in Dai et al., 2000)). One SNP (rs926103), located in exon 3, leads to a non-synonymous change at amino acid position 52 (serine (S) to asparagine (N) substitution). The five selected tagging SNPs were genotyped in all available Norwegian MS cases and controls. The non-
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Fig. 1. Allele distribution of the GA repeat polymorphism in the SH2D2A promoter gene in a newly collected Norwegian cohort of 311 MS patients and 277 controls.
synonymous SNP in exon 3, rs926103, was also genotyped in the Danish, Swedish and Finnish cases and controls and in an additional 1027 newly collected Norwegian controls. The SNPs were genotyped by TaqMan technology (Assay on demand) (C_16069698_10 = rs2768764, C_3178900_10 = rs1800600, C_7480922_20 = rs916203, C_7480910_10 = rs909200, C_16069690_10 = rs2768766) on an ABI Prism® 7000 analyzer. The success rates for genotyping of the SNPs were above 95% in all sample sets, notably above 99% in the Norwegian case– control cohort. 2.3. Statistical analysis Genetic power calculations were done at http://pngu.mgh. harvard.edu/~purcell/cgi-bin/cc2.cgi. Hardy–Weinberg equili-
brium was calculated using PEDSTATS (Wigginton and Abecasis, 2005), and all markers were in Hardy–Weinberg equilibrium in both cases and controls in all cohorts. The allele frequencies were compared between patients and controls, and odds ratios (OR) and P values were estimated using the Public Domain Software for Epidemiology and Disease Surveillance EPI Info Version 5.01b (Center of Disease Control, Epidemiology Program Office, Atlanta, GA, USA). Haplotype estimations were calculated using the program UNPHASED version 3.0.3. (Dudbridge, 2003). LD and r2 patterns between the markers were generated in Haploview version 3.32. (Barrett et al., 2005). The Breslow–Day test and Cochran–Mantel–Haenszel test were performed by using PLINK version 1.02 (Purcell et al., 2007). Clinical correlations and logistic regression analyses were performed using SPSS for Windows Version 14.0 (www. spss.com). P values below 0.05 in the initial analyses were ultimately corrected ad modum Bonferroni, by multiplying with the number of comparisons performed (n = 10, i.e. the 6 loci and 4 haplotypes considered), the exception being P values resulting from Cochran–Mantel–Haenszel test which are usually not corrected. 3. Results 3.1. The GA16 allele of the SH2D2A promoter polymorphism is associated with MS among Norwegians The GA repeat polymorphism in the SH2D2A promoter was previously reported to be associated with MS, both in Nordic MS multiplex families and in a Norwegian case–control sample set (Dai et al., 2001). In order to evaluate this association further, we collected a new cohort of 311 MS patients and 277 controls and genotyped this new sample set for the SH2D2A GA repeat polymorphism. The distribution of the individual GA
Table 1 Allele frequencies for all markers studied in the SH2D2A gene among Norwegian cases and controls Marker
a
Polymorphism
Minor allele
GA repeat polymorphism
153599913– 153599948
GAn
e
Marker
a
Polymorphism
Minor allele
rs2768766 rs909200 rs926103 rs1800600 GA repeat polymorphism rs2768764 a b c d e f g
Position
Position
153588707 153591184 153598055 153598852 153599913– 153599948 153600001
GA16
b
b
n = 311
n = 277
0.236
f
Norwegian MS, replication data
All Norwegian MS cases
c
c
d Pnc value
0.197
1.3
1.0–1.7
0.1
f
c
c
d Pnc value
Norwegian controls, replication data
All Norwegian controls
n = 624
n = 562
OR
OR
95% CI
95% CI
C/T G/A G/A T/C GAn
T A g A C e GA16
0.278 0.223 0.367 0.336 0.235
0.259 0.208 0.332 0.287 0.196
1.1 1.1 1.2 1.3 1.3
0.9–1.3 0.9–1.4 1.0–1.4 1.1–1.5 1.0–1.6
0.3 0.4 0.08 0.01 0.02
A/T
T
0.255
0.224
1.2
1.0–1.5
0.08
From UCSC Assembly May 2004 (http://genome.ucsc.edu/index.html). A newly collected Norwegian MS case–control cohort, not reported before. Odds ratio with 95% confidence interval. P value, not corrected for number of comparisons. GA16 allele for the GA repeat polymorphism in the SH2D2A promoter gene region. Include both reported previously (Dai et al., 2001) and new Norwegian MS cases and controls. In some populations the minor allele is found to be G (http://www.ncbi.nlm.nih.gov/sites/entrez).
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repeat alleles is depicted in Fig. 1. In accordance with our initial study where we observed a significant over-transmission of the GA16 allele in Nordic sib pairs (Dai et al., 2001), a higher frequency of the GA16 allele among Norwegian MS patients was observed also in the new case–control set (Table 1, upper part). The frequencies of the GA16 allele in the present and previously published Norwegian cases and controls were similar (0.236 vs. 0.234 and 0.197 vs. 0.194 respectively). We therefore chose to analyse the entire Norwegian case–control set as one single cohort in the remaining part of the study. In this combined Norwegian set, an association with the GA16 allele and MS was observed (OR 1.3 (1.0–1.6), Pnc = 0.02, Table 1, lower part). No differences in the distribution of GA repeat alleles were observed in HLA-DRB1⁎1501 positive versus negative Norwegian MS patients (data not shown).
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Also an independent set of 1027 Norwegian controls were genotyped for the rs926103 SNP. The genotype success rate was 99%, and the minor allele frequency in this additional Norwegian control cohort (0.331) was almost identical to the first Norwegian controls presented in this study (0.332). When this second Norwegian control data set was added to the Norwegian case– control cohort, the OR for MS conferred by the rs926103⁎A allele was found to be 1.2 (1.0–1.4, Pnc = 0.01) (data not shown).
3.2. Trend of association with SH2D2A SNPs in Norwegian MS cases and controls In order to further map the association conferred by the SH2D2A gene region, all available Norwegian MS patients and controls were genotyped for five selected SNPs tagging the SH2D2A gene (Fig. 2A). The D′ measurements showed a high degree of linkage disequilibrium (LD) between several of the tested SNPs, indicating a reduced haplotype diversity (Fig. 2B). As illustrated in Fig. 2C, all SNP combinations except one, showed a r2 below 0.8 in our data set. For all the five SNPs tested, the minor allele was found to be more common among MS patients than controls (Table 1, lower part). However only the rs1800600-C allele reached a Pnc value b 0.05 (0.336 vs. 0.287, OR 1.3 (1.1–1.5), Pnc = 0.01). 3.3. The GA16–rs926103⁎A haplotype is associated with MS among Norwegians The increased frequency of the minor alleles of the five tested SNPs could be due to LD with the GA16 allele. We therefore estimated haplotypes between the SNPs and the GA16 allele using the program UNPHASED. The GA repeat polymorphism was converted into a biallelic marker with GA16 as one allele, and all the other alleles grouped as the second allele. The minor alleles at four of the five loci (the exception being rs909200) were in strong LD with the GA16 allele (D′ values N 0.75). The five locus haplotype consisting of each of these minor alleles as well as the GA16 allele was still over-represented among MS patients (0.200 vs. 0.158, OR 1.3 (1.0–1.7), Pnc = 0.02). When GA16 negative patients and controls were compared, none of these SNPs showed any deviation in frequency between patients and controls (data not shown). Since the GA16 allele seemed to be in strong LD with four of the five SNPs tested (Fig. 2B and C), we chose to restrict our further analyses to haplotypes of the GA repeat polymorphism and rs926103, which encodes an amino acid change in the SH2D2A gene. The LD between the GA repeat polymorphism and rs926103 displayed a global D′ = 0.83. The haplotype comprising GA16 and rs926103⁎A, was positively associated in the Norwegian MS cohort (0.217 vs. 0.169, OR 1.4 (1.1–1.7), Pnc = 0.004, Pc = 0.04) (Table 2).
Fig. 2. An overview of the SH2D2A gene region, the location of genotyped markers and linkage disequilibrium patterns among the markers. A) New SNP markers were selected within a region two kb upstream and five kb downstream of the SH2D2A coding region, a total of 17.5 kb. In this figure the gene is read from right to left. The gene's exons are drawn in black. The five SNPs selected for genotyping are marked in black: One SNP (rs926103) is located in exon 3 and two SNPs are located in introns (rs1800600 in intron 2 and rs909200 in intron 7). The two remaining SNPs (rs2768766 and rs2768763) are located on each side outside the coding region. The genotyped GA repeat polymorphism in the promoter region is also marked in the figure. B) The LD patterns displayed as D′ values for the genotyped SNPs and the GA repeat polymorphism (the GA repeat polymorphism is converted into a biallelic marker with GA16 as one allele, and all the other alleles as the second allele) are shown in red. (Stronger colour indicates stronger D′. D′ values are in % of 1.00). C) The r2 values between the SNPs and the GA repeat polymorphism (the GA repeat polymorphism is converted into a biallelic marker with GA16 as one allele, and all the other alleles as the second allele) are shown on a black–white scale (the darker, the higher the r2 value. r2 values are given in % of 1.00). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 2 Haplotype frequencies for the GA repeat polymorphism and the SNP rs926103 for all the Norwegian MS cases and controls a GA– brs926103 haplotype
b c d e f
c
2n = 1248
2n = 1124
0.020 0.217 0.612 0.151
0.030 0.169 0.640 0.164
All Norwegian MS cases
f GA16–G GA16–A GAother–G GAother–A a
c
d
All Norwegian controls
d
OR
0.7 1.4 0.9 0.9
e
95% CI
Pnc value
0.4–1.3 1.1–1.7 0.8–1.1 0.7–1.1
0.2 0.004 0.2 0.4
GA repeat polymorphism in the promoter region grouped into GA16 and GAother allele before haplotype estimation. SNP rs926103 allele G and A. Include both reported previously (Dai et al., 2001) and new Norwegian MS cases and controls. Odds ratio with 95% confidence interval. P value, not corrected for number of comparisons. Reference haplotype is GA16–G.
3.4. No correlation between the SH2D2A polymorphisms and clinical presentation of MS For 571 of the Norwegian MS patients, sufficient clinical information was available to assess whether SH2D2A polymorphisms may influence the clinical presentation of the disease. However, logistic regression analyses revealed no correlations of the GA16 and rs926103⁎A alleles (Table 1) or haplotypes (Table 2) to gender, initial disease course and age at onset (data not shown). 3.5. The SH2D2A polymorphisms in Nordic populations Since we previously observed an association with the GA16 allele in Nordic sib pairs (Dai et al., 2001) we proceeded to examine the SH2D2A GA repeat and the rs926103 polymorphisms in Danish, Swedish and Finnish case–control sample sets. In all Nordic cohorts, the GA16 and the rs926103⁎A alleles were in strong LD with a global D′ ranging between 0.91 and 0.98 and r2 ranged between 0.45 and 0.48 (data not shown). In both the Danish and the Swedish data sets the GA16 allele was more common among patients than controls (Table 3, upper part), and the GA16–rs926103⁎A haplotype was more common in
Danish patients than controls (Table 3, lower part). However these differences did not reach statistical significance. The Danish MS patients and controls carried the GA16 allele and the rs926103⁎A allele at frequencies similar to that observed among the Norwegian patients and controls (Table 3, upper part), whereas the GA16 allele and the rs926103⁎A allele was higher among the Swedish and Finnish controls than among the Norwegian controls (Table 3, upper part). A Breslow–Day test showed however no significant heterogeneity across the populations for the two tested SH2D2A polymorphisms (P = 0.09 for the GA repeat and P = 0.4 for rs926103). Using the Cochran– Mantel–Haenszel test, that controls for the potential confounding due to the population variability, the average OR for the GA16 allele across the four populations was calculated to be 1.15 (1.04– 1.28) and P = 0.007. The average OR for the rs926103⁎A allele, however, did not reach statistical significance (OR 1.0 (0.94– 1.13) and P = 0.5). When the original Norwegian data set was excluded from the combined analysis, the average OR for the GA16 allele was OR = 1.10 (0.99–1.24 and P = 0.09). Further analysis of only the Scandinavian replication set (Norwegian, Danish and Swedish samples) revealed an average OR for the GA16 allele of 1.13 (1.0–1.28, P = 0.05).
Table 3 Allele- and haplotype frequencies for the GA repeat in the promoter region and SNP rs926103 in the SH2D2A gene for all typed MS cases and controls Marker
Position
Polymorphism Minor allele
n = 562
n = 620 n = 553
n = 644
n = 649
n = 240 n = 240
0.235
0.196
0.238
0.207
0.261
0.248
0.216
0.236
d
0.367
0.332
0.354
0.350
0.377
0.395
0.383
0.395
GA16–A
0.217
0.169
0.228
0.200
0.240
0.248
0.218
0.234
GAother–A 0.151
0.164
0.126
0.150
0.140
0.143
0.164
0.158
c
rs926103
GA–rs926103 haplotype
a b c d
G/A
Norwegian aNorwegian Danish Danish Swedish Swedish Finnish Finnish MS cases controls MS controls MS controls MS controls cases cases cases n = 624
GA repeat 153599913– GAn polymorphism 153599948 153598055
a
GA16
A
Include both reported previously (Dai et al., 2001) and new Norwegian MS cases and controls. P value, not corrected for number of comparisons. GA16 allele for the GA repeat polymorphism in the SH2D2A promoter gene region. In some populations the minor allele is found to be G (http://www.ncbi.nlm.nih.gov/sites/entrez).
b
Pnc-value
PNorwegian = 0.02 PDanish = 0.07 Pall = 0.007 PNorwegian = 0.004 PDanish = 0.1
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4. Discussion The present replication study performed on 2128 Nordic MS patients and 2004 Nordic unrelated controls shows that the SH2D2A gene may contribute to susceptibility to MS. In a recently published review by Plenge and Rioux (2006) the authors discussed the statistical power for a replication cohort, and pointed out that the original exploratory study often over-estimates the true magnitude of the genetic effect (‘Winner's Curse’ phenomenon). This means that replication studies often need to test more samples than the original study to detect an association. In the present study, we were able to include 311 new Norwegian MS patients and 277 new controls, which is a case–control sample set of similar size as the original one (Dai et al., 2001). The new Norwegian data set may therefore have been too small to be able to significantly replicate the previously reported association. Accordingly, we did not replicate our original finding that homozygous GA13 and/or GA16 genotypes were increased in Norwegian MS patients. In the new similar sized Norwegian data set, only the GA16 allele but not the rarer GA13 allele still showed increased allele frequency in patients compared to controls. In our original study, a significant over-transmission of the GA16 but not the GA13 allele was observed in Nordic sib pair families. We therefore also included Danish, Swedish and Finnish MS patients and controls in the present replication analysis. The Finnish population, especially the geographical subisolates, have been regarded as having a specific genetic structure characterised by the effect of multiple bottle necks and has often been considered nonhomogenous with other Nordic populations (Service et al., 2006). The Breslow–Day test however, ruled out the existence of significant heterogeneity across the four Nordic populations, irrespective of whether the Finnish samples were included or not. The statistical power of the combined material to detect at the P = 0.05 level a risk allele with similar population frequency as the GA16 allele among Norwegians (i.e. 0.19, Dai et al., 2001) and conferring an OR of 1.2, was found to be 75%, indicating that the combined material included in the present study is relatively well powered to detect an association with SH2D2A to MS. The finding of a significant Cochran–Mantel–Haenszel test generating OR of 1.15 across the four populations for the GA16 allele supports this notion. The Finnish data set might represent a population genetically different from the other Scandinavian populations (Service et al., 2006). Therefore, the Scandinavian replication set (Danish, Swedish and the new Norwegian samples only) was also analysed separately, and then an average OR of 1.13 (1.0–1.28) at the P = 0.05 level was observed for the GA16 allele. Of note is that in order to have 80% power to detect a risk allele similar to the SH2D2A GA16 allele (frequency 0.19, OR = 1.15 and P =0.05) approximately 4000 cases and controls are needed, and to reach P = 0.001, a total of 9000 cases and controls are needed. As many as 63 SNPs have been reported in the SH2D2A region targeted in this study. Thus, we might have missed the true causal variant displaying the strongest and most consistent association across all populations. However, the two polymorphisms focused on in this study, may well have biological significance and a possible direct effect on disease susceptibility conferred by the SH2D2A gene region. The GA repeat polymorphism in the
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SH2D2A promoter influences the expression of the SH2D2A gene (Dai et al., 2001). The rs926103⁎A allele encodes an asparagine at position amino acid 52 in TSAd, whereas the rs926103⁎G allele encodes the amino acid serine. It is presently unknown how this polymorphism could alter the function of TSAd. An intriguing possibility is that the serine in position 52 is a phosphorylation site that is lost in TSAd encoded by the rs926103⁎A allele. Since the publication of our first studies on SH2D2A association in autoimmune diseases (Dai et al., 2001; Smerdel et al., 2004), it has become clear that the SH2D2A gene is located very close to the neurotrophic tyrosine kinase receptor, type 1 (NTRK1) gene (also called TRKA) encoding nerve growth factor receptor A. In fact the first exon of NTRK1 is encoded within the second intron of the SH2D2A gene. SH2D2A is transcribed from the minus strand, the NTRK1 gene from the plus strand. One possibility which has to be explored is therefore that the observed association with the SH2D2A polymorphisms may be explained by effects of NTRK1 expression or function in MS. In conclusion, our study supports the notion that the SH2D2A gene region may contribute to the susceptibility to MS. However, additional replication studies in larger populations are needed, and the possible underlying mechanisms need to be further studied. Acknowledgements We warmly thank all individuals participating in this study. This study has been supported by the Medical Research Curriculum at the University of Oslo, The Research Council of Norway, Eastern Norway Regional Health Authority, VIRUUS committee at the Ullevål University Hospital, Odd Fellow MS society, Medinnova SF at Rikshospitalet, The Multiple Sclerosis Society of Norway, Fritz and Inger Nilsen's Fund, Inger R. Haldorsen's Fund, stud. med. Morten Dedekam Harboe's Fund and The Danish Multiple Sclerosis Society (Scleroseforeningen). The Norwegian Bone Marrow Donor Registry is thanked for collaboration in the establishment of the Norwegian control material and collaboration on HLA typing. The excellent technical assistance of Ingebjørg K. Heitman, Monica Hals, Wenche Hamang Scheel, Tone Aarskaug and Bente Woldseth, as well as technical staff at the Institute of Immunology, Rikshospitalet, is warmly appreciated. The statistical guidance of Thore Egeland for regression analyses is also acknowledged. And finally, the members of the “Nordic MS Genetic Network” are thanked for their contribution to this paper. References Barrett, J.C., Fry, B., Maller, J., Daly, M.J., 2005. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics 21 (2), 263–265. Dai, K.Z., Vergnaud, G., Ando, A., Inoko, H., Spurkland, A., 2000. The SH2D2A gene encoding the T-cell-specific adapter protein (TSAd) is localized centromeric to the CD1 gene cluster on human chromosome 1. Immunogenetics 51 (3), 179–185. Dai, K.Z., Harbo, H.F., Celius, E.G., Oturai, A., Sorensen, P.S., Ryder, L.P., Datta, P., Svejgaard, A., Hillert, J., Fredrikson, S., Sandberg-Wollheim, M., Laaksonen, M., Myhr, K.M., Nyland, H., Vartdal, F., Spurkland, A., 2001. The T cell regulator gene SH2D2A contributes to the genetic susceptibility of multiple sclerosis. Genes Immun. 2 (5), 263–268.
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Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M.A., Bender, D., Maller, J., Sklar, P., de Bakker, P.I., Daly, M.J., Sham, P.C., 2007. PLINK: A tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81 (3), 559–575. Rajagopal, K., Sommers, C.L., Decker, D.C., Mitchell, E.O., Korthauer, U., Sperling, A.I., Kozak, C.A., Love, P.E., Bluestone, J.A., 1999. RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation. J. Exp. Med. 190 (11), 1657–1668. Sawcer, S., Ban, M., Maranian, M., Yeo, T.W., Compston, A., Kirby, A., Daly, M.J., De Jager, P.L., Walsh, E., Lander, E.S., Rioux, J.D., Hafler, D.A., Ivinson, A., Rimmler, J., Gregory, S.G., Schmidt, S., Pericak-Vance, M.A., Akesson, E., Hillert, J., Datta, P., Oturai, A., Ryder, L.P., Harbo, H.F., Spurkland, A., Myhr, K.M., Laaksonen, M., Booth, D., Heard, R., Stewart, G., Lincoln, R., Barcellos, L.F., Hauser, S.L., Oksenberg, J.R., Kenealy, S.J., Haines, J.L., 2005. A high-density screen for linkage in multiple sclerosis. Am. J. Hum. Genet. 77 (3), 454–467. Service, S., DeYoung, J., Karayiorgou, M., Roos, J.L., Pretorious, H., Bedoya, G., Ospina, J., Ruiz-Linares, A., Macedo, A., Palha, J.A., Heutink, P., Aulchenko, Y., Oostra, B., van, D.C., Jarvelin, M.R., Varilo, T., Peddle, L., Rahman, P., Piras, G., Monne, M., Murray, S., Galver, L., Peltonen, L., Sabatti, C., Collins, A., Freimer, N., 2006. Magnitude and distribution of linkage disequilibrium in population isolates and implications for genomewide association studies. Nat. Genet. 38 (5), 556–560. Smerdel, A., Dai, K.Z., Lorentzen, A.R., Flato, B., Maslinski, S., Thorsby, E., Forre, O., Spurkland, A., 2004. Genetic association between juvenile rheumatoid arthritis and polymorphism in the SH2D2A gene. Genes Immun. 5 (4), 310–312. Spurkland, A., Brinchmann, J.E., Markussen, G., Pedeutour, F., Munthe, E., Lea, T., Vartdal, F., Aasheim, H.C., 1998. Molecular cloning of a T cellspecific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites. J. Biol. Chem. 273 (8), 4539–4546. Sundvold, V., Torgersen, K.M., Post, N.H., Marti, F., King, P.D., Rottingen, J. A., Spurkland, A., Lea, T., 2000. T cell-specific adapter protein inhibits T cell activation by modulating Lck activity. J. Immunol. 165 (6), 2927–2931. Wigginton, J.E., Abecasis, G.R., 2005. PEDSTATS: descriptive statistics, graphics and quality assessment for gene mapping data. Bioinformatics 21 (16), 3445–3447. Wu, L.W., Mayo, L.D., Dunbar, J.D., Kessler, K.M., Ozes, O.N., Warren, R.S., Donner, D.B., 2000. VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor. J. Biol. Chem. 275 (9), 6059–6062. Zucchelli, S., Holler, P., Yamagata, T., Roy, M., Benoist, C., Mathis, D., 2005. Defective central tolerance induction in NOD mice: Genomics and genetics. Immunity 22 (3), 385–396.
The SH2D2A gene and susceptibility to multiple sclerosis Åslaug R. Lorentzen a,c,⁎, Cathrine Smestad b , Benedicte A. Lie c , Annette B. Oturai d , Eva Åkesson e , Janna Saarela f , Kjell-Morten Myhr g,h , Frode Vartdal c,i , Elisabeth G. Celius b , Per S. Sørensen d , Jan Hillert e , Anne Spurkland j , Hanne F. Harbo b a
e
Department of Neurology, Faculty Division Ullevål University Hospital, University of Oslo, Oslo, Norway b Department of Neurology, Ullevål University Hospital, Oslo, Norway c Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway d Danish MS Research Center, Department of Neurology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Division of Neurology, Department of Clinical Neuroscience, Karolinska Institute at Karolinska University Hospital, Huddinge, Stockholm, Sweden f Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland g Department of Neurology, Haukeland University Hospital, Bergen, Norway h Department of Clinical Medicine, University of Bergen, Bergen, Norway i Faculty Division Rikshospitalet University Hospital, University of Oslo, Oslo, Norway j Institute of Basal Medical Science, University of Oslo, Oslo, Norway Received 10 January 2008; received in revised form 25 April 2008; accepted 29 April 2008
Abstract We previously reported an association between the SH2D2A gene encoding TSAd and multiple sclerosis (MS). Here a total of 2128 Nordic MS patients and 2004 controls were genotyped for the SH2D2A promoter GA repeat polymorphism and rs926103 encoding a serine to asparagine substitution at amino acid position 52 in TSAd. The GA16–rs926103⁎A haplotype was associated with MS in Norwegians (OR 1.4, P = 0.04). A similar trend was observed among Danes. In the independent Norwegian, Danish and Swedish sample sets the GA16 allele showed a combined OR of 1.13, P = 0.05. Thus, the present study shows that the SH2D2A gene may contribute to susceptibility to MS. © 2008 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; SH2D2A; Autoimmunity; Disease susceptibility; TSAd
1. Introduction Multiple sclerosis (MS) is presumed to be an autoimmune disease affecting the central nervous system. The disease leads to demyelination and axonal damage, and is one of the most common causes of neurological disability in young adults in western countries. Both environmental and genetic factors contribute to susceptibility and pathogenesis of the disease. Many potential susceptibility genes have been suggested, and until recently the human leukocyte antigen (HLA) class II gene
⁎ Corresponding author. Institute of Immunology, Rikshospitalet University Hospital, 0027 Oslo, Norway. Tel.: +47 23 07 35 00; fax: +47 23 07 35 10. E-mail address: [email protected] (Å.R. Lorentzen). 0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.04.037
region was the only region that was firmly established to be associated with MS (Jersild et al., 1973; Olerup and Hillert, 1991; Sawcer et al., 2005). However, the genetic susceptibility probably involves many different genes as postulated in the common allelic variants hypothesis (Dyment et al., 2004; Oksenberg and Barcellos, 2005). Recently, in a whole genome association study performed by typing 334 923 SNPs, an association with MS was observed for IL2RA and IL7RA in addition to genes in the HLA region (Hafler et al., 2007). We previously reported an association with MS to the SH2 domain protein 2A (SH2D2A) gene, which is located on chromosome 1q23.1. The gene encodes the T cell specific adaptor protein (TSAd), which is expressed in activated T cells (Spurkland et al., 1998; Sundvold et al., 2000), as well as in NK cells (Nejad et al., 2004) and endothelial cells (Matsumoto et al.,
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2005; Wu et al., 2000). Although the function of TSAd in T cells is not fully understood, TSAd is important for normal differentiation and activation of T cells. T cells from mice lacking the SH2D2A gene showed reduced capacity for proliferation and reduced production of the T helper cell type one (TH1) cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ) (Rajagopal et al., 1999). These mice spontaneously developed a lupus-like autoimmune disease (Drappa et al., 2003). The SH2D2A gene is located in a chromosomal region that has been implied in the genetic susceptibility to autoimmune diseases in mice (Johannesson et al., 2005). It is not yet known whether TSAd-knock-out mice are more prone to develop organ specific autoimmune diseases. However in the diabetes prone NOD mice, reduced SH2D2A expression may be linked to a defect in the induction of central tolerance (Zucchelli et al., 2005). Furthermore, a meta-analysis of published gene expression studies in MS patients revealed that T cells from MS patients display reduced expression of the SH2D2A gene (Fernald et al., 2005). A dinucleotide (GA) repeat polymorphism located in the SH2D2A promoter gene region, at position − 340 from the initiator ATG codon (Dai et al., 2000), has been found to be associated with susceptibility to MS in a Norwegian case– control cohort and in Nordic multiplex families (Dai et al., 2001). In Nordic MS sib pair families, the GA16 allele was overtransmitted, and among unrelated Norwegians the frequency of patients carrying homozygous GA13 and/or GA16 genotypes was higher than that observed in healthy controls. A similar association was observed in juvenile rheumatoid arthritis (Smerdel et al., 2004). Expression of TSAd in activated T cells was lower in healthy individuals carrying a homozygous SH2D2A GA13 and/or GA16 genotype (Dai et al., 2001), suggesting that the length variation of the promoter GA repeat could have a direct functional significance. Alternatively, the GA repeat polymorphism may be in linkage disequilibrium (LD) with some other disease promoting polymorphisms within the SH2D2A gene region. To further explore the contribution of the SH2D2A gene in MS, we fine mapped the SH2D2A gene in Norwegian MS cases and controls and extended our studies by including a large sample set of Nordic MS patients and controls. 2. Materials and methods 2.1. MS patients and controls In total, 2128 unrelated Nordic MS cases and 2004 healthy controls were included in the present study, from which DNA was extracted from blood samples using standard methods. This included 624 unrelated MS patients and 562 healthy controls from Norway. Some of these samples had previously been genotyped for the GA repeat polymorphism in the promoter region of the SH2D2A gene (MS n = 313 and control n = 277) (Dai et al., 2001). An additional 1027 newly collected Norwegian controls were genotyped for the SNP rs926103 only. All Norwegian MS patients were diagnosed as definite MS according to the Poser criteria (Poser et al., 1983) or the McDonald criteria (McDonald et al., 2001). The female:male
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ratio was 1.4:1, and the mean age at onset was 31.1 years (range 10–62 years). The initial disease course was relapsing remitting MS (RR-MS) in 82% of the patients and primary progressive MS (PP-MS) in 18% of the patients. The Norwegian control samples were all randomly collected among healthy blood donors recruited through the Norwegian Bone Marrow Donor Registry. The female:male ratio was 1.4:1 and 1.5:1 respectively in the two sets of controls. The remaining Nordic 1504 MS cases and 1442 controls were recruited from Denmark, Sweden and Finland. All the MS cases fulfilled either the definite MS criteria of Poser or the MS criteria of McDonald. In these MS samples the female:male ratio was 2.6:1, the mean age at onset was 31.8 years (range 11– 63 years), and 91.4% were classified initially as RR-MS. The control samples from the same three Nordic countries had a female:male ratio at 0.9:1. The regional Ethics Committees approved the protocol, and informed consent was obtained from each individual. 2.2. Marker selection and genotyping The GA repeat polymorphism in the promoter region of the SH2D2A gene was amplified from all available samples (Norwegian, Danish, Swedish and Finnish cases and controls) as previously described (Dai et al., 2000, 2001). The microsatellite alleles were identified by fragment length analysis on an ABI Prism® 377XL sequencer or ABI Prism® 3730 DNA sequencer (Applied Biosystems, Foster city, CA, USA). Data were analysed using GeneMapper® version 3.5 or 3.7 or Gene Scan® 2.1 and the Genotyper® 2.0 software. The success rates for genotyping of the GA repeat polymorphism were above 94.2% for all case–control cohorts. Single nucleotide polymorphisms (SNPs) were selected within a 17 566 base pair (bp) region, stretching from two kb upstream to five kb downstream of the SH2D2A coding region. In the target region between 37 and 63 SNPs were reported in the HapMap (HapMap Data Rel 20/phaseII Jan 06, on NCBI B35 assembly, dbSNP b 125) (http://www.hapmap.org/index.html), UCSC (Assembly May 2004) (http://genome.ucsc.edu/index. html), Entrez SNP (http://www.ncbi.nlm.nih.gov/) or Ensembl (http://www.ensembl.org/index.html) databases. Among these, 47 SNPs had allele frequencies or heterozygosity data available in the databases. By using the Haploview tagging tool (r2 N 0.8, minor allele frequency (MAF) N 0.1) five SNPs were selected to tag the SH2D2A region (Imported HapMap data in Haploview was used). None of the selected SNPs displayed a MAF below 0.10 in the database. When tagging the SH2D2A gene region for haplotypes, the r2 between the selected SNPs was estimated to be below 0.80. Two of the chosen SNPs were previously genotyped in a limited number of the Norwegian MS patients and healthy controls (rs2768764 in the SH2D2A promoter (− 446 in Dai et al., 2001) and rs1800600 in intron 2 (IVS2 + 21 in Dai et al., 2000)). One SNP (rs926103), located in exon 3, leads to a non-synonymous change at amino acid position 52 (serine (S) to asparagine (N) substitution). The five selected tagging SNPs were genotyped in all available Norwegian MS cases and controls. The non-
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Fig. 1. Allele distribution of the GA repeat polymorphism in the SH2D2A promoter gene in a newly collected Norwegian cohort of 311 MS patients and 277 controls.
synonymous SNP in exon 3, rs926103, was also genotyped in the Danish, Swedish and Finnish cases and controls and in an additional 1027 newly collected Norwegian controls. The SNPs were genotyped by TaqMan technology (Assay on demand) (C_16069698_10 = rs2768764, C_3178900_10 = rs1800600, C_7480922_20 = rs916203, C_7480910_10 = rs909200, C_16069690_10 = rs2768766) on an ABI Prism® 7000 analyzer. The success rates for genotyping of the SNPs were above 95% in all sample sets, notably above 99% in the Norwegian case– control cohort. 2.3. Statistical analysis Genetic power calculations were done at http://pngu.mgh. harvard.edu/~purcell/cgi-bin/cc2.cgi. Hardy–Weinberg equili-
brium was calculated using PEDSTATS (Wigginton and Abecasis, 2005), and all markers were in Hardy–Weinberg equilibrium in both cases and controls in all cohorts. The allele frequencies were compared between patients and controls, and odds ratios (OR) and P values were estimated using the Public Domain Software for Epidemiology and Disease Surveillance EPI Info Version 5.01b (Center of Disease Control, Epidemiology Program Office, Atlanta, GA, USA). Haplotype estimations were calculated using the program UNPHASED version 3.0.3. (Dudbridge, 2003). LD and r2 patterns between the markers were generated in Haploview version 3.32. (Barrett et al., 2005). The Breslow–Day test and Cochran–Mantel–Haenszel test were performed by using PLINK version 1.02 (Purcell et al., 2007). Clinical correlations and logistic regression analyses were performed using SPSS for Windows Version 14.0 (www. spss.com). P values below 0.05 in the initial analyses were ultimately corrected ad modum Bonferroni, by multiplying with the number of comparisons performed (n = 10, i.e. the 6 loci and 4 haplotypes considered), the exception being P values resulting from Cochran–Mantel–Haenszel test which are usually not corrected. 3. Results 3.1. The GA16 allele of the SH2D2A promoter polymorphism is associated with MS among Norwegians The GA repeat polymorphism in the SH2D2A promoter was previously reported to be associated with MS, both in Nordic MS multiplex families and in a Norwegian case–control sample set (Dai et al., 2001). In order to evaluate this association further, we collected a new cohort of 311 MS patients and 277 controls and genotyped this new sample set for the SH2D2A GA repeat polymorphism. The distribution of the individual GA
Table 1 Allele frequencies for all markers studied in the SH2D2A gene among Norwegian cases and controls Marker
a
Polymorphism
Minor allele
GA repeat polymorphism
153599913– 153599948
GAn
e
Marker
a
Polymorphism
Minor allele
rs2768766 rs909200 rs926103 rs1800600 GA repeat polymorphism rs2768764 a b c d e f g
Position
Position
153588707 153591184 153598055 153598852 153599913– 153599948 153600001
GA16
b
b
n = 311
n = 277
0.236
f
Norwegian MS, replication data
All Norwegian MS cases
c
c
d Pnc value
0.197
1.3
1.0–1.7
0.1
f
c
c
d Pnc value
Norwegian controls, replication data
All Norwegian controls
n = 624
n = 562
OR
OR
95% CI
95% CI
C/T G/A G/A T/C GAn
T A g A C e GA16
0.278 0.223 0.367 0.336 0.235
0.259 0.208 0.332 0.287 0.196
1.1 1.1 1.2 1.3 1.3
0.9–1.3 0.9–1.4 1.0–1.4 1.1–1.5 1.0–1.6
0.3 0.4 0.08 0.01 0.02
A/T
T
0.255
0.224
1.2
1.0–1.5
0.08
From UCSC Assembly May 2004 (http://genome.ucsc.edu/index.html). A newly collected Norwegian MS case–control cohort, not reported before. Odds ratio with 95% confidence interval. P value, not corrected for number of comparisons. GA16 allele for the GA repeat polymorphism in the SH2D2A promoter gene region. Include both reported previously (Dai et al., 2001) and new Norwegian MS cases and controls. In some populations the minor allele is found to be G (http://www.ncbi.nlm.nih.gov/sites/entrez).
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repeat alleles is depicted in Fig. 1. In accordance with our initial study where we observed a significant over-transmission of the GA16 allele in Nordic sib pairs (Dai et al., 2001), a higher frequency of the GA16 allele among Norwegian MS patients was observed also in the new case–control set (Table 1, upper part). The frequencies of the GA16 allele in the present and previously published Norwegian cases and controls were similar (0.236 vs. 0.234 and 0.197 vs. 0.194 respectively). We therefore chose to analyse the entire Norwegian case–control set as one single cohort in the remaining part of the study. In this combined Norwegian set, an association with the GA16 allele and MS was observed (OR 1.3 (1.0–1.6), Pnc = 0.02, Table 1, lower part). No differences in the distribution of GA repeat alleles were observed in HLA-DRB1⁎1501 positive versus negative Norwegian MS patients (data not shown).
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Also an independent set of 1027 Norwegian controls were genotyped for the rs926103 SNP. The genotype success rate was 99%, and the minor allele frequency in this additional Norwegian control cohort (0.331) was almost identical to the first Norwegian controls presented in this study (0.332). When this second Norwegian control data set was added to the Norwegian case– control cohort, the OR for MS conferred by the rs926103⁎A allele was found to be 1.2 (1.0–1.4, Pnc = 0.01) (data not shown).
3.2. Trend of association with SH2D2A SNPs in Norwegian MS cases and controls In order to further map the association conferred by the SH2D2A gene region, all available Norwegian MS patients and controls were genotyped for five selected SNPs tagging the SH2D2A gene (Fig. 2A). The D′ measurements showed a high degree of linkage disequilibrium (LD) between several of the tested SNPs, indicating a reduced haplotype diversity (Fig. 2B). As illustrated in Fig. 2C, all SNP combinations except one, showed a r2 below 0.8 in our data set. For all the five SNPs tested, the minor allele was found to be more common among MS patients than controls (Table 1, lower part). However only the rs1800600-C allele reached a Pnc value b 0.05 (0.336 vs. 0.287, OR 1.3 (1.1–1.5), Pnc = 0.01). 3.3. The GA16–rs926103⁎A haplotype is associated with MS among Norwegians The increased frequency of the minor alleles of the five tested SNPs could be due to LD with the GA16 allele. We therefore estimated haplotypes between the SNPs and the GA16 allele using the program UNPHASED. The GA repeat polymorphism was converted into a biallelic marker with GA16 as one allele, and all the other alleles grouped as the second allele. The minor alleles at four of the five loci (the exception being rs909200) were in strong LD with the GA16 allele (D′ values N 0.75). The five locus haplotype consisting of each of these minor alleles as well as the GA16 allele was still over-represented among MS patients (0.200 vs. 0.158, OR 1.3 (1.0–1.7), Pnc = 0.02). When GA16 negative patients and controls were compared, none of these SNPs showed any deviation in frequency between patients and controls (data not shown). Since the GA16 allele seemed to be in strong LD with four of the five SNPs tested (Fig. 2B and C), we chose to restrict our further analyses to haplotypes of the GA repeat polymorphism and rs926103, which encodes an amino acid change in the SH2D2A gene. The LD between the GA repeat polymorphism and rs926103 displayed a global D′ = 0.83. The haplotype comprising GA16 and rs926103⁎A, was positively associated in the Norwegian MS cohort (0.217 vs. 0.169, OR 1.4 (1.1–1.7), Pnc = 0.004, Pc = 0.04) (Table 2).
Fig. 2. An overview of the SH2D2A gene region, the location of genotyped markers and linkage disequilibrium patterns among the markers. A) New SNP markers were selected within a region two kb upstream and five kb downstream of the SH2D2A coding region, a total of 17.5 kb. In this figure the gene is read from right to left. The gene's exons are drawn in black. The five SNPs selected for genotyping are marked in black: One SNP (rs926103) is located in exon 3 and two SNPs are located in introns (rs1800600 in intron 2 and rs909200 in intron 7). The two remaining SNPs (rs2768766 and rs2768763) are located on each side outside the coding region. The genotyped GA repeat polymorphism in the promoter region is also marked in the figure. B) The LD patterns displayed as D′ values for the genotyped SNPs and the GA repeat polymorphism (the GA repeat polymorphism is converted into a biallelic marker with GA16 as one allele, and all the other alleles as the second allele) are shown in red. (Stronger colour indicates stronger D′. D′ values are in % of 1.00). C) The r2 values between the SNPs and the GA repeat polymorphism (the GA repeat polymorphism is converted into a biallelic marker with GA16 as one allele, and all the other alleles as the second allele) are shown on a black–white scale (the darker, the higher the r2 value. r2 values are given in % of 1.00). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 2 Haplotype frequencies for the GA repeat polymorphism and the SNP rs926103 for all the Norwegian MS cases and controls a GA– brs926103 haplotype
b c d e f
c
2n = 1248
2n = 1124
0.020 0.217 0.612 0.151
0.030 0.169 0.640 0.164
All Norwegian MS cases
f GA16–G GA16–A GAother–G GAother–A a
c
d
All Norwegian controls
d
OR
0.7 1.4 0.9 0.9
e
95% CI
Pnc value
0.4–1.3 1.1–1.7 0.8–1.1 0.7–1.1
0.2 0.004 0.2 0.4
GA repeat polymorphism in the promoter region grouped into GA16 and GAother allele before haplotype estimation. SNP rs926103 allele G and A. Include both reported previously (Dai et al., 2001) and new Norwegian MS cases and controls. Odds ratio with 95% confidence interval. P value, not corrected for number of comparisons. Reference haplotype is GA16–G.
3.4. No correlation between the SH2D2A polymorphisms and clinical presentation of MS For 571 of the Norwegian MS patients, sufficient clinical information was available to assess whether SH2D2A polymorphisms may influence the clinical presentation of the disease. However, logistic regression analyses revealed no correlations of the GA16 and rs926103⁎A alleles (Table 1) or haplotypes (Table 2) to gender, initial disease course and age at onset (data not shown). 3.5. The SH2D2A polymorphisms in Nordic populations Since we previously observed an association with the GA16 allele in Nordic sib pairs (Dai et al., 2001) we proceeded to examine the SH2D2A GA repeat and the rs926103 polymorphisms in Danish, Swedish and Finnish case–control sample sets. In all Nordic cohorts, the GA16 and the rs926103⁎A alleles were in strong LD with a global D′ ranging between 0.91 and 0.98 and r2 ranged between 0.45 and 0.48 (data not shown). In both the Danish and the Swedish data sets the GA16 allele was more common among patients than controls (Table 3, upper part), and the GA16–rs926103⁎A haplotype was more common in
Danish patients than controls (Table 3, lower part). However these differences did not reach statistical significance. The Danish MS patients and controls carried the GA16 allele and the rs926103⁎A allele at frequencies similar to that observed among the Norwegian patients and controls (Table 3, upper part), whereas the GA16 allele and the rs926103⁎A allele was higher among the Swedish and Finnish controls than among the Norwegian controls (Table 3, upper part). A Breslow–Day test showed however no significant heterogeneity across the populations for the two tested SH2D2A polymorphisms (P = 0.09 for the GA repeat and P = 0.4 for rs926103). Using the Cochran– Mantel–Haenszel test, that controls for the potential confounding due to the population variability, the average OR for the GA16 allele across the four populations was calculated to be 1.15 (1.04– 1.28) and P = 0.007. The average OR for the rs926103⁎A allele, however, did not reach statistical significance (OR 1.0 (0.94– 1.13) and P = 0.5). When the original Norwegian data set was excluded from the combined analysis, the average OR for the GA16 allele was OR = 1.10 (0.99–1.24 and P = 0.09). Further analysis of only the Scandinavian replication set (Norwegian, Danish and Swedish samples) revealed an average OR for the GA16 allele of 1.13 (1.0–1.28, P = 0.05).
Table 3 Allele- and haplotype frequencies for the GA repeat in the promoter region and SNP rs926103 in the SH2D2A gene for all typed MS cases and controls Marker
Position
Polymorphism Minor allele
n = 562
n = 620 n = 553
n = 644
n = 649
n = 240 n = 240
0.235
0.196
0.238
0.207
0.261
0.248
0.216
0.236
d
0.367
0.332
0.354
0.350
0.377
0.395
0.383
0.395
GA16–A
0.217
0.169
0.228
0.200
0.240
0.248
0.218
0.234
GAother–A 0.151
0.164
0.126
0.150
0.140
0.143
0.164
0.158
c
rs926103
GA–rs926103 haplotype
a b c d
G/A
Norwegian aNorwegian Danish Danish Swedish Swedish Finnish Finnish MS cases controls MS controls MS controls MS controls cases cases cases n = 624
GA repeat 153599913– GAn polymorphism 153599948 153598055
a
GA16
A
Include both reported previously (Dai et al., 2001) and new Norwegian MS cases and controls. P value, not corrected for number of comparisons. GA16 allele for the GA repeat polymorphism in the SH2D2A promoter gene region. In some populations the minor allele is found to be G (http://www.ncbi.nlm.nih.gov/sites/entrez).
b
Pnc-value
PNorwegian = 0.02 PDanish = 0.07 Pall = 0.007 PNorwegian = 0.004 PDanish = 0.1
Å.R. Lorentzen et al. / Journal of Neuroimmunology 197 (2008) 152–158
4. Discussion The present replication study performed on 2128 Nordic MS patients and 2004 Nordic unrelated controls shows that the SH2D2A gene may contribute to susceptibility to MS. In a recently published review by Plenge and Rioux (2006) the authors discussed the statistical power for a replication cohort, and pointed out that the original exploratory study often over-estimates the true magnitude of the genetic effect (‘Winner's Curse’ phenomenon). This means that replication studies often need to test more samples than the original study to detect an association. In the present study, we were able to include 311 new Norwegian MS patients and 277 new controls, which is a case–control sample set of similar size as the original one (Dai et al., 2001). The new Norwegian data set may therefore have been too small to be able to significantly replicate the previously reported association. Accordingly, we did not replicate our original finding that homozygous GA13 and/or GA16 genotypes were increased in Norwegian MS patients. In the new similar sized Norwegian data set, only the GA16 allele but not the rarer GA13 allele still showed increased allele frequency in patients compared to controls. In our original study, a significant over-transmission of the GA16 but not the GA13 allele was observed in Nordic sib pair families. We therefore also included Danish, Swedish and Finnish MS patients and controls in the present replication analysis. The Finnish population, especially the geographical subisolates, have been regarded as having a specific genetic structure characterised by the effect of multiple bottle necks and has often been considered nonhomogenous with other Nordic populations (Service et al., 2006). The Breslow–Day test however, ruled out the existence of significant heterogeneity across the four Nordic populations, irrespective of whether the Finnish samples were included or not. The statistical power of the combined material to detect at the P = 0.05 level a risk allele with similar population frequency as the GA16 allele among Norwegians (i.e. 0.19, Dai et al., 2001) and conferring an OR of 1.2, was found to be 75%, indicating that the combined material included in the present study is relatively well powered to detect an association with SH2D2A to MS. The finding of a significant Cochran–Mantel–Haenszel test generating OR of 1.15 across the four populations for the GA16 allele supports this notion. The Finnish data set might represent a population genetically different from the other Scandinavian populations (Service et al., 2006). Therefore, the Scandinavian replication set (Danish, Swedish and the new Norwegian samples only) was also analysed separately, and then an average OR of 1.13 (1.0–1.28) at the P = 0.05 level was observed for the GA16 allele. Of note is that in order to have 80% power to detect a risk allele similar to the SH2D2A GA16 allele (frequency 0.19, OR = 1.15 and P =0.05) approximately 4000 cases and controls are needed, and to reach P = 0.001, a total of 9000 cases and controls are needed. As many as 63 SNPs have been reported in the SH2D2A region targeted in this study. Thus, we might have missed the true causal variant displaying the strongest and most consistent association across all populations. However, the two polymorphisms focused on in this study, may well have biological significance and a possible direct effect on disease susceptibility conferred by the SH2D2A gene region. The GA repeat polymorphism in the
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SH2D2A promoter influences the expression of the SH2D2A gene (Dai et al., 2001). The rs926103⁎A allele encodes an asparagine at position amino acid 52 in TSAd, whereas the rs926103⁎G allele encodes the amino acid serine. It is presently unknown how this polymorphism could alter the function of TSAd. An intriguing possibility is that the serine in position 52 is a phosphorylation site that is lost in TSAd encoded by the rs926103⁎A allele. Since the publication of our first studies on SH2D2A association in autoimmune diseases (Dai et al., 2001; Smerdel et al., 2004), it has become clear that the SH2D2A gene is located very close to the neurotrophic tyrosine kinase receptor, type 1 (NTRK1) gene (also called TRKA) encoding nerve growth factor receptor A. In fact the first exon of NTRK1 is encoded within the second intron of the SH2D2A gene. SH2D2A is transcribed from the minus strand, the NTRK1 gene from the plus strand. One possibility which has to be explored is therefore that the observed association with the SH2D2A polymorphisms may be explained by effects of NTRK1 expression or function in MS. In conclusion, our study supports the notion that the SH2D2A gene region may contribute to the susceptibility to MS. However, additional replication studies in larger populations are needed, and the possible underlying mechanisms need to be further studied. Acknowledgements We warmly thank all individuals participating in this study. This study has been supported by the Medical Research Curriculum at the University of Oslo, The Research Council of Norway, Eastern Norway Regional Health Authority, VIRUUS committee at the Ullevål University Hospital, Odd Fellow MS society, Medinnova SF at Rikshospitalet, The Multiple Sclerosis Society of Norway, Fritz and Inger Nilsen's Fund, Inger R. Haldorsen's Fund, stud. med. Morten Dedekam Harboe's Fund and The Danish Multiple Sclerosis Society (Scleroseforeningen). The Norwegian Bone Marrow Donor Registry is thanked for collaboration in the establishment of the Norwegian control material and collaboration on HLA typing. The excellent technical assistance of Ingebjørg K. Heitman, Monica Hals, Wenche Hamang Scheel, Tone Aarskaug and Bente Woldseth, as well as technical staff at the Institute of Immunology, Rikshospitalet, is warmly appreciated. The statistical guidance of Thore Egeland for regression analyses is also acknowledged. And finally, the members of the “Nordic MS Genetic Network” are thanked for their contribution to this paper. References Barrett, J.C., Fry, B., Maller, J., Daly, M.J., 2005. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics 21 (2), 263–265. Dai, K.Z., Vergnaud, G., Ando, A., Inoko, H., Spurkland, A., 2000. The SH2D2A gene encoding the T-cell-specific adapter protein (TSAd) is localized centromeric to the CD1 gene cluster on human chromosome 1. Immunogenetics 51 (3), 179–185. Dai, K.Z., Harbo, H.F., Celius, E.G., Oturai, A., Sorensen, P.S., Ryder, L.P., Datta, P., Svejgaard, A., Hillert, J., Fredrikson, S., Sandberg-Wollheim, M., Laaksonen, M., Myhr, K.M., Nyland, H., Vartdal, F., Spurkland, A., 2001. The T cell regulator gene SH2D2A contributes to the genetic susceptibility of multiple sclerosis. Genes Immun. 2 (5), 263–268.
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