Is Glutathione Peroxidase-1 (GPX1) the Relevant Gene?

SHORT COMMUNICATION Inﬂammatory bowel disease (IBD) locus 12: is glutathione peroxidase-1 (GPX1) the relevant gene?

F Häuser1, H Rossmann1, D Laubert-Reh2, PS Wild2,3,4, T Zeller5,6, C Müller5, S Neuwirth1, S Blankenberg5,6 and KJ Lackner1

Genome-wide association studies have identified and repeatedly confirmed the association of rs3197999 in MST1 with inflammatory bowel disease (IBD). However, the underlying pathophysiology remains unclear. rs3197999 is a non-synonymous single-nucleotide polymorphism which modifies the function of macrophage stimulating protein-1 (MST1). We show by haplotyping that rs3197999 is in linkage disequilibrium with rs1050450 in GPX1, with almost complete cosegregation of the minor alleles. As shown by immunoassay, rs3197999 influences the MST-1 level in serum. But also rs1050450 causes an amino acid exchange in glutathione peroxidase 1 (GPx-1) and reduced activity of this antioxidant enzyme. The association of GPx deficiency and IBD in mice was already shown. We propose that GPx-1 is a better candidate than MST1 for the pathophysiologic link between IBD locus 12 and IBD.

Genes and Immunity (2015) 16, 571–575; doi:10.1038/gene.2015.35; published online 10 September 2015

INTRODUCTION the true causative variant has also been discussed by the authors Today more than 160 genetic susceptibility loci are known for of the numerous genome-wide association studies (GWAS) and inflammatory bowel diseases (Crohn´s disease and ulcerative meta-analyses describing the association of rs3197999 with IBD 1,3,9,10 colitis, IBD). The association of some single-nucleotide polymorph- and/or PSC. But the question was not answered. The isms (SNP) with IBD was replicated convincingly several times, for currently available studies are purely descriptive (that is, several example rs3197999 (MST1) and rs9858542 (BSN).1–4 Both variants or all potential candidate genes within IBD locus 12 are listed, are located in IBD locus 12 a 10.7 Mb spanning region located on among them in several cases: BSN, MST1 and GPX1) with a clear chromosome 3 (3p21; Figure 1), show similar minor allele focus on MST1 due to the fact that rs3197999 represents a frequencies (MAF) (for example, rs9858542: 0.1953, rs3197999: missense mutation affecting protein function. 0.1919 [dbSNP]), and cosegregate in the same haplotype (http:// In this situation, we set out to search for further functionally hapmap.ncbi.nlm.nih.gov). Notably a large number of further SNPs relevant SNPs in the region 3p21 (Table 1). Among other SNPs we with comparable MAF are located in IBD locus 12 (Table 1) and are identified rs1050450 (c. 599C4T), a polymorphism in the presumably linked to rs3197999, but for the majority linkage has glutathione peroxidase-1 (GPX1) gene as a promising candidate. not yet been formally established. rs3197999, one of the best replicated SNPs in IBD and primary sclerosing cholangitis, is a variant in the macrophage stimulating RESULTS AND DISCUSSION protein (MST1/MSP/HGFL) gene. The MST1 which is the ligand of GPX1 SNP rs1050450 has not been reported to be associated with the receptor tyrosine kinase RON (recepteur dórigine nantais) was IBD or to cosegregate with the MST1 SNP rs3197999 to date. It found to induce a variety of cellular responses, for example, results in an amino acid substitution (p. Pro200Leu); the Leu migration, proliferation and adhesion.5,6 rs3197999 (c.2107C4T) is variant shows a reduced peroxidase activity in erythrocytes and a common SNP in MST1, resulting in the substitution of an the activity is less responsive to increasing selenium blood arginine by a cysteine at position 703 of MST1. In vitro the SNP levels.11,12 GPx-1 is a selenoprotein with antioxidant and anti- causes a ‘gain of function’; the mutant protein induces migration inflammatory functions. As expected because of their small and proliferation in macrophage-like cells more efficiently than physical distance on chromosome 3 (327 kb) and their similar the wild-type protein without changing the ability of binding its MAF (MAF rs3197999: 0.1919, MAF rs1050450: 0.2175 [dbSNP]), we receptor RON.7,8 observed that rs3197999 (in MST1) and rs1050450 (in GPX1) are Since rs3197999 is at the same time associated to IBD and does closely linked (Figure 1). Our haplotype analysis revealed that modify macrophage function, while rs9858542 (BSN) is a synon- there is almost complete cosegregation of the minor alleles ymous variant, rs3197999 has been regarded as the prime (Table 2). By a systematic genome-wide search for haplotypic cis candidate for a pathophysiologic link between IBD locus 12 and eQTL effects of rs3197999 in monocytes based on 1374 individuals IBD. Nevertheless, the question whether rs3197999 itself is of the population-based Gutenberg Health Study of the University causally related to IBD or PSC or is in linkage disequilibrium with Medical Center Mainz, we found the mRNA expression of five

1Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Mainz, Mainz, Germany; 2Department of Medicine II, Preventive Cardiology and Preventive Medicine, University Medical Center Mainz, Mainz, Germany; 3Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany; 4German Center for Cardiovascular Research (DZHK), Partner Site RhineMain, Mainz, Germany; 5Department of General and Interventional Cardiology, University Heart Center Hamburg, Hamburg, Germany and 6German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, Hamburg, Germany. Correspondence: Dr H Rossmann, Institute of Clinical Chemistry and Laboratory Medicine, University Medical Centre Mainz, Langenbeckstrasse 1, Mainz 55131, Germany. E-mail: [email protected] Received 9 June 2015; revised 17 July 2015; accepted 27 July 2015; published online 10 September 2015 IBD locus 12: is GPX1 the relevant gene? F Häuser et al 572

Figure 1. Scheme of IBD locus 12 (3p21). Chromosomal localization of the genes, distances between genes and SNPs as given by the UCSC Genome Brower (http://ucscbrowser.genap.ca) and the NCBI dbSNP (http://www.ncbi.nlm.nih.gov/SNP).

genes significantly associated with the rs3197999 genotype consequence of the disease.15 (4) GPx-1 activity decreases in Se- (Supplementary Table S1). All of them are located within an deficiency.12 Furthermore GPx-1 activity in carriers of the 800-kb region of 3p21 (Figure 1), and among them is GPX1 (eQTL; rs1050450 minor allele (C/T, T/T) does not increase with rising effect estimate 0.0712, P-valueo0.001, false discovery rate 0.01). Se-concentrations to that extent observed in C/C individuals;16,17 in a cohort of 4900 probands of the Gutenberg Health Study these data suggest us that Se-deficiency is more relevant in (Figure 2a). Median GPx-1 activity in erythrocytes of individuals, carriers of the minor allele. (5) Several case reports and small which are homozygous for the MST1 SNP rs3197999 (T/T), is studies suggest a beneficial effect of Se-supplementation in the significantly lower (59.8 U g− 1 Hb; Po0.001) than in individuals prevention or treatment of IBD, even though randomized with heterozygous (C/T; 62.4 U g − 1 Hb) or homozygous C alleles controlled studies are still lacking. (C/C; 63.9 U g − 1 Hb). Hence the GPx-1 effect of rs3197999 (MST1) Though the association of rs3197999 in MST1 with IBD was first is consistent with the effect of rs1050450 (GPX1) published published in 2008,1 replicated and included in several meta- previously (C/C vs C/T minus 3.3%, C/C vs T/T minus 7.7%).11 For analyses, it is still unclear, which variant in IBD locus 12 causes this MST1 serum concentration, the effect is even more pronounced association. Of course there are more genetic variants in IBD locus (Figure 2b): Median serum MST1 levels of individuals with 12 and though most of them are for several reasons not likely to homozygous minor alleles (rs3197999: T/T) are significantly lower be involved in pathophysiological processes (for example, (129.82 ng ml − 1, Po0.001) than levels of those with heterozygous synonymous SNPs, far intronic SNPs, gene function without (C/T: 193.64 ng ml − 1, Po0.001) or homozygous C alleles (C/C: obvious link to IBD), there still remain several SNPs with possible − 266.36 ng ml 1). Our mRNA expression and protein data suggest a significance for IBD (Table 1). For example, a variable number of common regulation of MST1 and GPX1. However, we cannot alanine repeats (Ala7/6/5; Ala6: rs139760138, Ala5 rs17838762) in completely rule out that the two SNPs have independent effects the GPx-1 amino terminus has been shown to interact with on their genes and gene products. rs1050450 and affect the protein's cellular localization and There are different reasons why association studies were not function.18 One could speculate that stratifying our data by this focused on rs1050450: first of all this SNP were not analyzed. additional variation might even result in a more pronounced Genome-wide association studies results were largely generated effect of the rs1050450 genotype. by arrays, which detect a defined panel of SNPs. rs1050450 was In summary, we show by linkage and functional analysis that not part of these panels (the Illumina Exome Chip is the first available genome-wide association studies data showing an platform analyzing rs3197999 and rs1050450 in one array; increased risk of IBD in carriers of the minor allele of rs3197999 Illumina, Inc; San Diego, CA, USA). More importantly, no linkage of MST1 apply as well to the minor allele of rs1050450 of GPX1. data for rs1050450 with rs3197999 is available in the HapMap Since a wealth of experimental and clinical data support a role for project’s database. GPx-1 in the pathogenesis of IBD, we propose that GPX1 is the Since rs1050450 is so closely linked to rs3197999 it is safe to most relevant gene within IBD locus 12. Further work is needed to assume that its minor allele is associated with an increased IBD- elucidate the precise pathogenic mechanisms. risk. At the same time there is strong evidence for a pathophysiologic role of GPx-1 in IBD: (1) glutathione peroxidase deficiency has been shown to lead to severe IBD in GPX1 − / −/ GPX2− / − MATERIALS AND METHODS double knockout mice.13 (2) In an experimental colitis model in Genotyping mice selenium (Se)-supplementation significantly reduced colitis- Genomic DNA of 4900 male and female participants aged associated inflammation.14 (3) Se-deficiency is much more 35–75 years of the Gutenberg Health Study of the University common in IBD patients than in healthy individuals and is Medical Center Mainz19 was isolated by phenol/chloroform thought to be involved in IBD pathophysiology rather than to be a extraction and genotyped for rs3197999 and a randomly

Table 1. RefSeq genes and SNPs as listed by the UCSC Genome Browser for IBD locus 3p21

genes within the ‘800 kb region’

3p21 ‘800 kb region‘ USP4 GPX1 NICN1 APEH MST1 RBM6

RefSeq genes in region 469 49 region size (kb) 10700 800 63 1.2 7 9.5 4.8 137.2 all SNPs 208734 15510 common SNPs (MAF 41%) 36616 2054 154 5 11 11 31 379 Common SNPs (MAF 41%) Nonsense 12 3 1 Frameshift 5 1 splice-5 1 splice-3 3 1 1 Stop-loss 1 cds-indel 4 Missense 435 44 5 3 8 1 ncRNA 254 10 Untranslated-5 130 10 1 1 Untranslated-3 502 29 3 1 Near-gene-5 1972 143 6 4 1 6 5 Near-gene-3 343 13 2 1 Intron 20546 1501 135 6 7 8 370 Coding-synon 265 34 1 2 7 2 Unknown 12143 264 2 1 SNPs, MAF = 15–35% Nonsense 2 Frameshift splice-5 1 splice-3 1 Stop-loss cds-indel Missense 63 4 1* 2# ncRNA 56 2 Untranslated-5 28 2 1 Untranslated-3 91 8 1 Near-gene-5 381 25 1 2 1 1 Near-gene-3 59 2 Intron 3803 320 20 1 2 2 62 Coding-synon 42 12 1 3 1 Unknown 2652 57 1 #rs3197999 *rs1050450 #rs397831743 Abbreviations: MAF, minor allele frequencies; SNP, single-nucleotide polymorphism. For the MST1 ﬂanking genes USP4, GPX1, NICN1, APEH and RBM6, which show rs3197999 (MST1) genotype-dependent mRNA expression, and the ‘800-kb region’, which is deﬁned by these genes (see Figure 1). The lower part of the table displays only those SNPs with a MAF of 0.14–0.35, which are potentially linked to rs3197999. Furthermore common SNPs and SNPs with a MAF of 0.14–0.35 were grouped according to their predicted function by a Perl script. Functional categories were adopted from the UCSC genome browser (http://ucscbrowser.genap.ca). Abbreviations used as described in the NCBI Handbook (http://www.ncbi.nlm.nih.gov/books/NBK21101/).

GGC-3′(rs3197999), 5′-TGCCCCTACGCAGGTACAG-3′ and 5′-Biotin- Table 2. Linkage of rs3197999 (MST1) and rs1050450 (GPX1) in 90% of GCCAAGCAGCCGGGGTAG- 3′(rs1050450), respectively. The the analyzed cases amplification protocol was as follows: initial incubation at MST1 GPx1 Number of individuals per Sum of % 95 °C for 5 min, followed by a 10 cycles touchdown protocol rs3197999 rs1050450 genotype individuals (95 °C for 60 s; 60455 °C for 30 s; and 72 °C for 30 s), 35 amplification cycles (95 °C for 60 s; 55 °C for 30 s; and C/C C/C 57 91.9 72 °C for 30 s) and a final elongation at 72 °C for 7 min. C/C C/T 5 8.1 The amplicons were sequenced by the PSQ 96MA instrument C/C T/T 0 62 0 fi T/T C/C 0 0 (Qiagen; Hilden, Germany) using a speci c sequencing primer: T/T C/T 4 5.6 5′-GTATGCGCAAGGTCC-3′ (rs3197999) and 5′-GCCCTGCTGT T/T T/T 67 71 94.4 CTCAAG-3′ (rs1050450).

Analysis of the MST1 protein and GPx-1 activity selected subgroup of 133 probands for rs1050450. Both SNPs The GPx-1 activity was determined in whole blood of the were determined by pyrosequencing. Genomic DNA was 4900 probands by the RANSEL glutathione peroxidase assay ampliﬁed using the following primer pairs 5′-CTGCTG (Randox Laboratories, Crumlin, UK),based on the method GGTCCTGGAAGGAAT-3′ and 5′-Biotin-CCTCCCCAAGGCATAT described by Paglia and Valentine.20 The assay was adapted to

Figure 2. GPx-1 activity in red blood cells and serum MST1 concentration depend on MST1 genotype (rs3197999). (a) GPx-1 activity of 4900 individuals grouped by rs3197999 genotype is presented by a box and whisker plot. One GPx Unit (U) is deﬁned as the enzyme activity which converts 1 μmol NADPH to NADP+ per min at 25 °C. (b) MST1 serum concentration of 4900 individuals grouped by rs3197999 genotype is shown by a box and whisker plot.

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Supplementary Information accompanies this paper on Genes and Immunity website (http://www.nature.com/gene)