Studies using whole genome scanning have highlighted chromosomes 3, 7, 12 and 16. A 32 base-pair deletion in the CC-chemokine receptor-5 gene ... the co-receptor that allows entry of the human immunodeficiency virus type. 1 (HIV-1) into ...
Genes and Immunity (2001) 2, 114–116 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene
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The CC chemokine receptor 5 ⌬32 mutation is not associated with inflammatory bowel disease (IBD) in NE England A Craggs1, M Welfare1, PT Donaldson1–2 and JC Mansfield1 1
Department of Gastroenterology, 2Centre for Liver Research, School of Clinical Medical Sciences, 4th Floor, William Leech Building, The Medical School, Framlington Place, University of Newcastle-upon-Tyne NE2 4HH, UK
Inflammatory bowel disease (IBD) is a group of chronic inflammatory diseases of the gastrointestinal tract of unknown aetiology. Evidence of abnormalities in immune regulation and cytokine production in patients with IBD has led to investigations of various immuno-regulatory genes as potential candidate susceptibility loci. Studies using whole genome scanning have highlighted chromosomes 3, 7, 12 and 16. A 32 base-pair deletion in the CC-chemokine receptor-5 gene (CCR5-⌬32, chromosome 3p21.3) has been associated with susceptibility to IBD. We have investigated CCR5 as a candidate susceptibility gene in 350 patients (251 with ulcerative colitis and 99 with Crohn’s disease) and 103 controls using polymerase chain reaction. There were no significant differences in the distribution of CCR5 genotypes or frequencies comparing patients and controls, or associations with extent of colitis. In contrast to preliminary data, these findings suggest no evidence for involvement of this mutation in susceptibility/resistance or disease progression in IBD. Genes and Immunity (2001) 2, 114–116. Keywords: inflammatory bowel disease (IBD); ulcerative colitis (UC); Crohn’s disease (CD); CCR5⌬-32 mutation; genetic polymorphism
Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract of unknown aetiology, comprising ulcerative colitis (UC) and Crohn’s disease (CD). Genetic factors are well recognised and are of considerable importance in the aetiology of both UC and CD.1 Evidence from family clustering2,3 and increased concordance for IBD in monozygotic vs dizygotic twins4 has provided the impetus to search genetic susceptibility loci. Studies using whole genome scanning have identified possible linkage on chromosomes 3, 7, 12 and 16,5 but have failed to identify any specific genes responsible. Population based case-control studies which have previously focused on the major histocompatibility complex (MHC) on chromosome 6p21.3 are currently gaining popularity as an alternative/complimentary approach to whole genome scanning. Short-listed candidates in IBD include genes which encode various immuno-regulatory proteins and there has been particular interest in cytokines,6 especially the pro-inflammatory cytokines tumour necrosis factor-alpha (TNFA) and the interleukin-1 (IL-
Correspondence: Alison Craggs, Department of Gastroenterology, School of Clinical Medical Sciences, 4th Floor, William Leech Building, The Medical School, Framlington Place, University of Newcastle-upon-Tyne NE2 4HH, UK. E-mail: Alison.Craggs얀ncl.ac.uk This study was supported by the Special Trustees of the Royal Victoria Infirmary (RVI), Newcastle upon Tyne. Received 21 October 2000; revised 3 December 2000; accepted 19 December 2000
1) gene family (IL-1A, IL-1B and IL-1RN). However, the majority of these studies have been disappointing. Recent interest has focussed on the role of chemokines, which promote subset-specific leukocyte recruitment to tissue and are upregulated during chronic inflammation and may play a crucial role in determining which leukocyte subsets are recruited to the bowel.7 One particular member of the chemokine receptor family; CCR5, has been identified as the co-receptor that allows entry of the human immunodeficiency virus type 1 (HIV-1) into human CD4-positive T cells.8 The gene encoding CCR5 is located on chromosome 3p21.3 adjacent to one of several putative IBD susceptibility loci identified from whole genome scanning.5 CCR5 is expressed on monocytes, macrophages, memory T cells and dendritic cells, and is believed to play an important role in the chemokinesis of these cell populations in vitro and in vivo in response to RANTES, MIP-1␣ and MIP1.9,10 A 32 base-pair deletion in the CCR5 gene referred to as (CCR5-⌬32) appears to protect individuals from infection with the M-tropic strain of HIV.11 Thus homozygotes for the ⌬32 deletion (a natural knock-out) are protected from infection despite repeated exposure12,13 and the development of clinical stages of AIDS is prolonged in heterozygotes.14,15 Genetic polymorphism within chemokine receptors may also be important for the establishment and progression of inflammatory process in IBD. In addition to IBD, recent studies have linked CCR5-⌬32 with protection from rheumatoid arthritis16 and a lower rate of disease recurrence in multiple sclerosis.17,18 Studies in chil-
CCR5 ⌬32 gene mutation in IBD A Craggs et al
dren with insulin dependent diabetes mellitus (IDDM) suggest that CCR2 gene may be a candidate in IDDM.19 A recent report has suggested a putative link between ulcerative colitis and a 32 base-pair mutation in the CCR5 gene (CCR5-⌬32).20 We have now evaluated the CCR5-⌬32 gene mutation in a total of 350 patients with IBD comprising: 107 UC and 99 CD (series 1) and 144 UC (series 2) from two NE hospitals and 103 healthy controls, and sought genetic associations with susceptibility/resistance to both CD and UC and correlation with extent of colitis. Overall there were no significant differences in the distribution of the CCR5 genotypes and allele frequencies in either series of patients when compared to controls (Tables 1 and 2). UC patients were subdivided into those with pan-colitis and either LS or distal disease, again no significant differences in genotype distribution were seen. The CCR5 genotype ⌬32, ⌬32 was seen in only one out of the 107 series one UC patients and one out of the 144 series two UC patients, and in none of the 103 controls. Comparing CD patients with controls showed a lower frequency of heterozygotes (14% vs 22%), though this difference was not statistically significant. However, the frequency of the ⌬32 allele was similar in both CD patients and controls (10% vs 11%). The variation in genotype distribution may be accounted for through moderate increase in ⌬32 homozygotes, 3% vs none of the controls, though this is not significant. The present study is entirely negative, failing to supTable 1 CCR5-⌬32 genotype distribution in ulcerative colitis (UC) and Crohn’s disease (CD) Wt/⌬32
Population
No.
Wt/Wt
Controls
103
80 (78%)
23 (22%)
Series 1 CD UC Pan-colitis Distal colitis Not known
99 107 47 58 2
82 84 34 48
14 22 12 10
Series 2 UC
144
(83%) (78.5%) (72%) (83%) 2
113 (78.5%)
(14%) (20.5%) (26%) (17%) 0
30 (21%)
⌬32/⌬32 0 3 (3%) 1 (1%) 1 (2%) 0 0 1 (0.5%)
The CCR5 gene mutation was amplified by polymerase chain reaction (PCR) in a 25-l reaction volume with the primers: 5⬘GTCTTCATTACACCTGCAGCTC-3⬘ forward and 5⬘GTGAAGA TAAGCCTCACAGCC-3⬘ reverse (Oswel, Southampton, UK). Template DNA (1 l) was amplified in reaction mixtures containing 200 m each of dATP, dCTP, dGTP and dTTP (Amersham Pharmacia-Biotech, St Albans, UK), 10 mm tris-HCl pH 8.3, 50 mm KCl, 0.01% gelatin., 1 U Taq Polymerase (AB Gene, Surrey, UK) and 0.5 m of each primer on a Perkin-Elmer GeneAmp 9700 System. The amplification conditions were as follows: initial denaturation at 94°C for 5 min followed by 30 cycles of, 94°C for 1 min, 66°C for 1 min, followed by 72°C for 1 min and a final extension at 72°C for 10 min. Following amplification the PCR-amplicons were visualised on 2.5% (w/v) agarose gels with ethidium bromide staining. Genotypes were assigned based on fragment size as follows: 198 bp only for wild-type homozygous (wt, wt), 198 bp plus 166 bp for wildtype heterozygous (wt, ⌬32) and 166 bp only for homozygous ⌬32 mutation (⌬32, ⌬32). Band sizes were compared using a commercially available size marker, Hinc II digested x174 phage DNA (AB Gene, Surrey, UK).
Table 2 CCR5-⌬32 allele frequencies in ulcerative colitis (UC) and Crohn’s disease (CD) Population
No.
Wt
⌬32
Controls
206
183 (89%)
23 (11%)
Series 1 CD UC Pan-colitis
198 214 94
178 (90%) 190 (89%) 80 (85%)
20 (10%) 24 (11%) 14 (15%)
Distal colitis Not known
116 4
106 (91%) 4
10 (9%) 0
Series 2 UC
288
256 (8%)
32 (11%)
115
Genotype and allele distribution of patients and controls was compared using 2 and Fisher’s exact probability tests with Bonferroni’s correction as appropriate. All analysis was performed using the EPISTAT statistical analysis programme (CDC-Atlanta Georgia, USA) on standard IBM-PC. Throughout the analysis series 1 patients was evaluated as a first UC set and the series 2 patients as a second UC set.
port any association between CCR5-⌬32 and IBD. This contrasts with a recent preliminary report in the UK suggesting positive association between CCR5-⌬32 and IBD: control data 13% vs UC 25% and CD 39% respectively for wt, ⌬32 heterozygotes. Our study, by comparison has a trend towards lower genotype frequency for heterozygotes than controls but found no increase in ⌬32 in IBD overall. The study of CCR5 as a potential candidate for IBD, although initially fully warranted both in terms of function and position is not the first positive genetic association study to be subsequently rejected upon further investigation. Replication of any initial genetic association is now increasingly recognised as an essential prerequisite for publication.21,22 The testing of more than one patient population serves as a means of replication for any finding and will reduce the likelihood of false positive associations in future studies, as well as spuriously negative results. A number of factors could explain the different results seen between our study and that of the preliminary report, there may be different genetic susceptibilities in familial and sporadic IBD. One of the strengths of the present study is that it was conducted in a stable population with minimal admixture. We also studied an unselected population containing a mixture of patients with and without a family history of IBD. The absence of an association between CCR5-⌬32 and IBD does not indicate that chemokines are unimportant in IBD, only that this particular mutation does not determine disease susceptibility or disease progression. A review of the literature suggests a complex interaction between CC-chemokine secretion and CC-chemokine receptor expression at immune activated sites during innate or adaptive immune response mediated by cytokine regulation. For example IL-13 and TNF-␣ can act as a potent inhibitory stimuli to CCR5 expression.23,24 IL-2 can also serve to downregulate CCR5 expression on macrophages, suggesting that T cell activation-induced IL-2 secretion can potentially reduce infection of target Genes and Immunity
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macrophages. IFN-␥ and IL-10 are implicated in increasing CCR5 expression on monocyte subsets. This negative study confirms the importance of replication of data in a second series. The genetic basis of IBD susceptibility remains largely unknown and further studies are necessary to identify other genes involved.
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