DNase1 has been implicated in a number of immune disorders and is an excellent candidate gene for Crohn's disease (CD). We investigated whether DNase1 SNPs rs1053874 and rs8176938 were associated with CD in a well-characterized New Zealand dataset consisting of 447 cases and 716 controls. Furthermore, we measured serum DNase1 activity levels in a number of CD patients and controls. We did not find any evidence of association for either DNase1 genetic variation or DNase1 activity levels with CD. The lack of association indicates that DNase1 does not play a significant role in predisposing to CD in the New Zealand population.
Deoxyribonuclease 1 (DNase1) is an endonuclease that facilitates chromatin breakdown. Deficiency in DNase1 and the resulting difficulty of removing DNA from nuclear antigens promote susceptibility to immune disorders such as systemic lupus erythematosus (SLE) [
Genetic variation in DNase1 has been associated with SLE [
We investigated whether DNase1 SNPs rs1053874 and rs8176938 were associated with CD in a well-characterized case-control New Zealand dataset. Furthermore, we measured serum DNase1 activity levels in a number of CD patients and controls.
A total of 1163 subjects from New Zealand were included in the study: 447 cases and 716 controls. All participants self-reported European ancestry.
Clinical records were analysed to confirm diagnosis, and IBD status was defined using standard diagnostic criteria [
Participants consented to collection of peripheral blood or a buccal swab for DNA extraction and genotyping, and DNA was extracted from the blood/buccal samples using Qiagen’s DNA extraction kit and following the manufacturer’s instructions.
The study was conducted under ethical protocol MEC/04/12/011, authorised through the New Zealand Multiregion Human Ethics Committee. All study subjects gave informed consent.
We searched for SNPs in the DNase1 gene in the hapmap database and identified 2 common (MAF > 5%) SNPs: rs1053874 and rs8176938. We genotyped these SNPs using the ABI TaqMan MGB diallelic discrimination system and predesigned assays on demand (Applied Biosystems, Melbourne, Australia). The polymerase chain reaction amplification was performed using the ABI Prism 7900 HT sequence-detector machine using the following conditions: 10 minutes at 95°C, followed by 40 cycles at 92°C for 15 seconds and 60°C for 1 minute. The allelic discrimination results were determined after the amplification by performing an endpoint read.
All sample plates contained cases, controls, blanks, hapmap, and duplicate samples. Quality control measures included independent double genotyping, blind to sample identity, and comparison of our hapmap sample genotypes to those in the hapmap database (
DNase1 activity levels were measured in the serum of 213 CD patients and 209 controls using a commercially available validated enzyme-linked immunosorbent assay (ELISA) kit (Orgentec, Germany), and the protocol was followed as described by the manufacturer. All samples were analyzed in duplicate. Activity levels were determined from a standard curve using a 4-parameter-fit with lin-log coordinates for optical density and concentration.
SNPs were tested for deviation from HWE in both cases and controls using a chi-square goodness-of-fit test. To determine if there were differences between cases and controls, genotype and allele frequencies for each SNP were analyzed by 2 × 3 and 2 × 2 Chi-square tables, respectively.
Genotype and phenotype associations were assessed by comparing allele frequencies between controls and patient subgroups defined using the clinical characteristics. These analyses were carried out using R (R: a language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL
Across-group comparisons of serum DNase 1 activity were tested using a generalized linear model for mean differences.
Both SNPs were in Hardy-Weinberg equilibrium. Neither was found to be associated with CD (Table
Genotype and allele counts (and frequencies) in CD patients and in controls.
SNP | Case | Control | Case | Control | ||||
---|---|---|---|---|---|---|---|---|
rs1053874 | A/A | 40 (9.0) | 65 (9.2) | 0.56 | A | 262 (29.5) | 442 (31.2) | 0.40 |
A/G | 182 (41.0) | 312 (44.0) | G | 626 (70.5) | 976 (68.8) | |||
G/G | 222 (50.0) | 332 (46.8) | ||||||
rs8176938 | C/C | 410 (91.7) | 644 (91.2) | 0.83 | C | 855 (95.6) | 1348 (95.5) | 0.85 |
C/G | 35 (7.8) | 60 (8.5) | G | 39 (4.4) | 64 (4.5) | |||
G/G | 2 (0.4) | 2 (0.3) |
Relationships between SNPs and subphenotypes.
rs1053874 (tested allele: G) | rs8176938 (tested allele: C) | ||||
OR (95% CI) | OR (95% CI) | ||||
Age at diagnosis | <17 | 0.86 (0.54–1.36) | 0.509 | 1.45 (0.45–4.60) | 0.533 |
17 to 40 | 1.02 (0.81–1.28) | 0.900 | 1.36 (0.80–2.33) | 0.259 | |
40< | 1.08 (0.72–1.62) | 0.707 | 0.86 (0.39–1.89) | 0.712 | |
CD behaviour | inflammatory | 1.12 (0.88–1.44) | 0.361 | 1.27 (0.72–2.22) | 0.411 |
stricturing | 0.78 (0.58–1.06) | 0.113 | 1.01 (0.53–1.95) | 0.969 | |
penetrating | 1.22 (0.74–2.00) | 0.433 | 2.20 (0.53–9.10) | 0.275 | |
CD location | Ileal | 0.96 (0.72–1.29) | 0.802 | 0.86 (0.49–1.52) | 0.605 |
Colonic | 1.11 (0.82–1.51) | 0.500 | 1.62 (0.74–3.54) | 0.229 | |
Ileocolonic | 0.95 (0.69–1.30) | 0.726 | 1.75 (0.75–4.10) | 0.195 | |
Other IBD family | Y | 0.89 (0.54–1.48) | 0.651 | 1.20 (0.37–3.89) | 0.759 |
Bowel resection | Y | 0.91 (0.68–1.21) | 0.517 | 1.43 (0.70–2.91) | 0.322 |
EIM | Y | 0.89 (0.61–1.32) | 0.568 | 0.90 (0.41–1.98) | 0.799 |
Perianal disease | Y | 1.33 (0.84–2.10) | 0.226 | — | |
Severity | Y | 1.00 (0.69–1.47) | 0.982 | 1.19 (0.51–2.78) | 0.695 |
Smoking | Y | 1.08 (0.74–1.58) | 0.677 | 0.95 (0.34–2.61) | 0.916 |
DNase1 activity levels were measured in the serum. Activity in the controls ranged from 43 to 100%. Activity in the cases ranged from 39 to 100%. The mean serum DNase1 activity for CD patients was 80% and that for the controls was also 80%. Thus, there was no difference in activity levels between cases and controls. We also compared DNase1 activity levels across the different CD phenotypes, but there were no significant findings (Table
DNase1 activity by subphenotype.
Estimate (95% CI) | |||
---|---|---|---|
Age at diagnosis | <17 | −0.23 (−4.09–3.64) | 0.908 |
17 to 40 | 1.51 (−0.75–3.78) | 0.190 | |
40< | −2.37 (−6.39–1.65) | 0.247 | |
CD behaviour | inflammatory | −0.16 (−2.66–2.35) | 0.903 |
stricturing | 1.96 (−1.01–4.93) | 0.195 | |
penetrating | 0.88 (−3.32–5.07) | 0.681 | |
CD location | Ileal | 0.10 (−2.76–2.97) | 0.943 |
Colonic | 1.29 (−1.61–4.19) | 0.382 | |
Ileocolonic | 0.38 (−2.69–3.44) | 0.809 | |
Other IBD family | Y | 0.50 (−4.56–5.57) | 0.846 |
Bowel resection | Y | 1.38 (−1.47–4.23) | 0.340 |
EIM | Y | −0.14 (−3.63–3.35) | 0.939 |
Perianal disease | Y | −0.83 (−4.79–3.14) | 0.681 |
severity | Y | 1.05 (−2.48–4.59) | 0.559 |
Smoking | Y | 1.01 (−3.46–5.65) | 0.633 |
DNase1 is known to be involved in a number of immune disorders, but research into its role in CD has been rare. There have been no previous studies investigating associations between genetic variation in DNase1 and CD, and only one published study examined DNase1 activity levels in CD.
We were unable to replicate the association reported in previous research in which DNase1 activity in CD patients was significantly lower than in healthy individuals [
As well as investigating DNase1 activity levels, we also undertook an analysis of genetic variation in DNase1 and compared the genotype and allele frequencies of 2 SNPs in CD patients and controls. Genetic variation in DNase1 has previously been associated with SLE [
To summarise, although DNase1 is a good candidate for CD, in the present study we failed to detect any significant associations. The absence of association between DNase1 and CD indicates that DNase1 does not play a significant role in predisposing to CD in the New Zealand population.
The authors would like to thank the many patients with IBD who participated in this study, and also the control subjects. Nutrigenomics New Zealand is collaboration between AgResearch Ltd., Plant & Food Research and The University of Auckland with funding through the Ministry of Science and Innovation (MSI).