Purpose
To evaluate the prevalence of immunologic and genetic markers in patients with idiopathic ocular inflammation and a family history of inflammatory bowel disease.
Design
Matched case-control study.
Methods
Patients with a diagnosis of idiopathic ocular inflammation and family history of inflammatory bowel disease who did not have inflammatory bowel disease themselves were identified and matched to control patients with idiopathic ocular inflammation. Serum was evaluated for immunologic markers using Prometheus IBD Serology 7. Genomic DNA was analyzed for single nucleotide polymorphisms (SNP) of the NOD2 gene associated with Crohn disease.
Results
Fifteen patients with idiopathic ocular inflammation and family history of inflammatory bowel disease were matched to 15 control patients based on age, sex, and race. Eight of 15 patients (53%) with a family history of inflammatory bowel disease had elevated p-ANCA antibody levels compared to 3 of 15 controls (20%) (1-sided P = .04) with a matched analysis odds ratio of 6.0 (1-sided P = .06). Four of 15 patients (27%) with family history of inflammatory bowel disease tested positive for immunologic markers predicting ulcerative colitis, while no control patients tested positive (1-sided P = .06). Carrier rates of NOD2 SNPs did not differ significantly between the test and control groups.
Conclusions
One-quarter of patients with idiopathic ocular inflammation and a family history of inflammatory bowel disease had immunologic markers predicting bowel disease, and one-half had elevated p-ANCA levels. Prometheus IBD Serology 7 may be useful in the evaluation of selected patients with unexplained uveitis.
Ocular inflammation is seen in 6% to 14% of patients with inflammatory bowel disease. Ocular involvement can include conjunctivitis, keratitis, scleritis, episcleritis, uveitis, optic neuritis, and, less commonly, retinal vasculitis, and can precede gastrointestinal disease. Uveitis is the most common ocular finding in patients with inflammatory bowel disease and is typically classified as chronic anterior uveitis.
We have previously reported that the prevalence of a family history of inflammatory bowel disease is 3-fold to 15-fold higher in patients with ocular inflammation than in the general population. Additionally, 74% of these patients were HLA-B27-negative, suggesting that HLA-B27 is not an adequate diagnostic marker for patients with only a family history of inflammatory bowel disease and idiopathic uveitis.
This study was conducted in order to identify immunologic markers specific to inflammatory bowel disease in a cohort of patients with idiopathic ocular inflammation and a family history of inflammatory bowel disease. Additionally, we assessed the presence or absence of single nucleotide polymorphisms (SNPs) in the NOD2 gene (also known as CARD15 ), which is known to be involved in the heritable aspect of Crohn disease.
Methods
This study was designed as a matched case-control study of patients diagnosed with idiopathic uveitis at the department of ophthalmology and visual sciences at the University of Illinois at Chicago (UIC). A retrospective chart review encompassing all patients seen at the UIC uveitis clinic between 1995 and 2010 was conducted to identify patients with a history of idiopathic uveitis. A diagnosis of idiopathic uveitis was made in patients with ocular inflammation in which the diagnostic examination did not result in a known etiology. Examination included rapid plasma reagin, fluorescent treponemal antibody absorption test, chest radiograph, angiotensin converting enzyme, and serum lysozyme, as well as other testing based on the patient’s history, review of systems, and examination findings. Patients with acute anterior uveitis were also tested for HLA-B27. Those with a positive result were excluded from the study.
Patients with idiopathic ocular inflammation and a family history of a first-, second-, or third-degree relative with inflammatory bowel disease without a personal history of inflammatory bowel disease were recruited and enrolled in this study. First-degree relatives were defined as parents, siblings, or children; second-degree relatives included aunts and uncles; and third-degree relatives included cousins or grandparents. Age-, sex-, and race-matched control patients were recruited with a diagnosis of idiopathic uveitis without a personal or family history of inflammatory bowel disease. Age match was defined as chronological age within 10 years of a test subject. Informed consent was obtained from all recruited participants. Clinical evaluation included a medical history, review of systems, and ophthalmic examination. Patients underwent standard venipuncture phlebotomy to obtain serum and anticoagulated whole blood samples.
Sample size estimates were based on prevalence studies of p-anti-neutrophil cytoplasmic antibodies (p-ANCA) and anti- Saccharomyces antibodies in inflammatory bowel disease patients. Studies report a range of 40% to 80% of ulcerative colitis patients testing positive for p-ANCA, while 60% to 70% of tested Crohn disease patients had elevated anti- Saccharomyces titers. First-degree relatives of those with ulcerative colitis have been reported to have p-ANCA prevalence rates of 15% to 30%, while anti- Saccharomyces antibody prevalence among first-degree relatives with Crohn disease ranges from 20% to 25%. Normal population controls show rates of 0% to 5%, while studies of “diseased” controls with undefined colitis reveal rates up to 10% for both p-ANCA and anti- Saccharomyces antibodies. As this study uses diseased controls, sample size calculations were based on an assumption of 10% prevalence among controls without a positive family history and assumed a prevalence of 25% among cases with positive family histories. Sample size calculations to detect an odds ratio of 5 with a study powered to 80% with a 1-way level of significance of 5% required 31 pairs (12 discordant pairs). However, given limited recruitment attributable to low disease prevalence, the study as presented was powered at 80% with a 1-way level of significance of 5% to detect an odds ratio (OR) of 8 based on the above assumptions of prevalence.
Immunologic Studies
A single vial of serum was sent to Prometheus Laboratories (San Diego, California, USA) for analysis using the Prometheus IBD Serology 7. Prometheus IBD Serology 7 assesses the following immunologic markers by enzyme-linked immunosorbent assay (ELISA): anti- Saccharomyces immunoglobulin (Ig) A, anti- Saccharomyces IgG, anti-OmpC IgA, anti-CBir1 antibodies, and p-ANCA antibodies, with additional testing for DNAse sensitivity and indirect immunofluorescence (IFA) perinuclear staining pattern. Inflammatory bowel disease–specific p-ANCA is determined by DNAse sensitivity and IFA perinuclear staining.
The results of these assays are imputed into a proprietary diagnostic algorithm allowing for a “laboratory-predicted serology” vs “laboratory-nonpredicted serology” result, assessing the risk of having either ulcerative colitis or Crohn disease. Prometheus IBD Serology 7 is marketed as having 74% sensitivity and 86% specificity for use as a diagnostic test for inflammatory bowel disease. The specificity for each individual bowel disease, ulcerative colitis and Crohn disease, is slightly higher, at 93% and 92%, respectively (unpublished internal data, Prometheus Labs, San Diego, California, USA).
Sequencing Analysis
Fresh, anticoagulated whole blood was sent to Oregon Health & Science University (OHSU), where it was processed to extract genomic DNA by standard procedures. Polymerase chain reaction (PCR) was performed using primers designed to amplify appropriate regions of exons 4, 8, and 11 of NOD2 . Direct sequencing of the PCR products was obtained in both directions. Four SNPs were genotyped: rs2066842 (C>T, encoding P268S, single-letter amino acid abbreviations, and position), rs2066844 (C>T, encoding R702W), rs2066845 (G>C, encoding G908R), and rs5743293 (a C insertion causing a frame shift resulting in premature termination: 1007fs).
Statistical Analysis
A matched analysis was performed using McNemar testing with exact P values calculated. One-sided P value testing was performed as the hypothesis tested was greater correlation of inflammatory bowel disease markers with those patients with a family history of disease. One-sided analysis was chosen a priori based on published data supporting the association of a family history of inflammatory bowel disease with the risk of development of ocular inflammation. Comparisons of proportions and matching characteristics were evaluated by Fisher exact testing. Analyses of NOD2 risk alleles comparing test vs control patient groups were performed with the Fisher exact test and the reported P values are uncorrected for multiple comparisons.
Results
Fifteen patients with a diagnosis of idiopathic uveitis and a family history of inflammatory bowel disease without a personal history of inflammatory bowel disease were included. Another 15 patients diagnosed with idiopathic uveitis without a family or personal history of inflammatory bowel disease were used as controls. Demographics of both groups are summarized in Table 1 . Fourteen patients in each group were of white descent and 1 patient was of African-American ancestry. The mean age of our test and control groups was similar at 35.8 years (range 8–61) and 35.4 years (range 16–58), respectively. The majority of patients in the test group (12 out of 15) had an inflammatory bowel disease–affected first-degree relative.
| With Inflammatory Bowel Disease Family History (Test) | Without Inflammatory Bowel Disease Family History (Control) | |
|---|---|---|
| Age, mean (range) | 35.8 years (8–61) | 35.4 years (16–58) |
| Sex (male:female) | 2:13 | 2:13 |
| Race (White:African American) | 14:1 | 14:1 |
| First-degree relative | N = 12 | — |
| Second-degree relative | N = 2 | — |
| Third-degree relative | N = 1 | — |
The type of uveitis represented in each group is presented in Table 2 . Although subjects were not matched as to disease location, there were no statistically significant differences between groups ( Table 2 ).
| With Inflammatory Bowel Disease Family History (Test), n (%) (N = 15) | Without Inflammatory Bowel Disease Family History (Control), n (%) N = 15) | P Value a | |
|---|---|---|---|
| Acute anterior uveitis | 4 (27%) | 1 (6%) | .16 |
| Chronic iridocyclitis | 3 (20%) | 7 (47%) | .12 |
| Intermediate uveitis | 6 (40%) | 2 (13%) | .11 |
| Scleritis/sclerokeratitis | 2 (13%) | 4 (27) | .32 |
| Posterior uveitis | 0 (0%) | 1 (6%) | .50 |
Of the 15 test patients, 9 (60%) had a family history of ulcerative colitis, while 6 (40%) had a family history of Crohn disease. Four of the 15 patients (27%) had serology predicting inflammatory bowel disease on the Prometheus IBD Serology 7, as shown in Table 3 . All 4 of these test patients were “UC-predicted,” meaning their results indicated positive ulcerative colitis immunologic markers. None of the control subjects had positive Prometheus IBD Serology 7; thus, an odds ratio could not be calculated. However, McNemar testing demonstrated a possible association (1-sided P = .063). Interestingly, 2 of the patients with predicted ulcerative colitis had a first- or second-degree relative with Crohn disease, while the other 2 had a first- or third-degree relative with ulcerative colitis. No patient developed ulcerative colitis during follow-up, although 3 of the 4 “UC-predicted” patients had follow-up of less than 1 year, while 1 patient has been followed for 5 years. None of the patients in this series developed Crohn disease, with follow-up ranging from less than 1 month to 5 years (median 1.75 years).
| With Inflammatory Bowel Disease Family History (Test) (N = 15) | Without Inflammatory Bowel Disease Family History (Control) (N = 15) | P Value a | |
|---|---|---|---|
| Predicted IBD-UC | 4 (27%) | 0 (0%) | .063 |
| Predicted IBD-CD | 0 (0%) | 0 (0%) | |
| IBD-specific pANCA | 8 (53%) | 3 (20%) | .063 |
| DNAse sensitivity | 4 (27%) | 0 (0%) | .063 |
| Anti-cBir-1 | 4 (27%) | 1 (6%) | .126 |
| Anti- Saccharomyces IgG OR IgA | 0 (0%) | 1 (6%) | .999 |
a One-sided exact P value using McNemar test (matched analysis).
Further analysis of individual immune markers revealed an association with p-ANCA antibodies. Eight of the 15 test patients (53%) had an elevated p-ANCA antibody ELISA compared to 3 of 15 control patients (20%) (1-sided P = .040). Matched analysis resulted in an OR = 6.0 (1-sided P = .063). All 4 test patients with UC-predicted results were positive for inflammatory bowel disease–specific p-ANCA, as determined by DNAse sensitivity and IFA perinuclear staining. No controls had positive inflammatory bowel disease–specific p-ANCA ( Table 3 ).
Anti- Saccharomyces serologies were negative in all test patients and were observed to be positive in only 1 control patient.
Ten test patients and 12 control patients were evaluated for 4 SNPs of the NOD2 gene. Three of these SNPs (R702W, G908R, and 1007fs) are highly associated with Crohn disease and 1 (P268S) has some evidence of disease association. All 4 variants encode changes to the resultant peptide. Our test population and control populations each demonstrated 1 individual who was positive for the R702W substitution, with all other patients exhibiting the major allele. Similarly, 1 test patient exhibited the SNP encoding G908R, while no control patients carried this variant. While not considered a major risk allele for Crohn disease, the SNP encoding P268S was also examined and found to be present in 4 individuals of the test cohort (1 was homozygous) and in 6 individuals of the control cohort. The distribution of each NOD2 SNP was not statistically different between the test and control groups ( Table 4 ). Furthermore, the overall minor allele frequencies of each SNP in the uveitis patients in this study are within reported ranges of other white cohorts (data not shown), so a skewed association with the uveitis phenotype appears to be unlikely.
