March 2021
Volume 62, Issue 3
Open Access
Cornea  |   March 2021
Innate and Adaptive Gene Single Nucleotide Polymorphisms Associated With Susceptibility of Severe Inflammatory Complications in Acanthamoeba Keratitis
Author Affiliations & Notes
  • Nicole A. Carnt
    School of Optometry and Vision Science, University of New South Wales (UNSW), Sydney, Australia
    Westmead Institute for Medical Research, Westmead, New South Wales, Australia
    University College London (UCL) Institute of Ophthalmology, London, United Kingdom
  • Ignatius Pang
    School of Biotechnology and Biomolecular Sciences, University of New South Wales (UNSW), Sydney, Australia
  • Kathryn P. Burdon
    Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia
  • Virginia Calder
    University College London (UCL) Institute of Ophthalmology, London, United Kingdom
  • John K. Dart
    University College London (UCL) Institute of Ophthalmology, London, United Kingdom
    Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
  • Dinesh Subedi
    School of Optometry and Vision Science, University of New South Wales (UNSW), Sydney, Australia
    School of Biological Sciences, Monash University, Clayton, Australia
  • Alison J. Hardcastle
    University College London (UCL) Institute of Ophthalmology, London, United Kingdom
  • Correspondence: Nicole Carnt, School of Optometry and Vision Science, University of New South Wales (UNSW), Level 3, Rupert Myers Building North Wing, Gate 14, Barker St, 2052, Sydney, Australia; n.carnt@unsw.edu.au
Investigative Ophthalmology & Visual Science March 2021, Vol.62, 33. doi:https://doi.org/10.1167/iovs.62.3.33
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      Nicole A. Carnt, Ignatius Pang, Kathryn P. Burdon, Virginia Calder, John K. Dart, Dinesh Subedi, Alison J. Hardcastle; Innate and Adaptive Gene Single Nucleotide Polymorphisms Associated With Susceptibility of Severe Inflammatory Complications in Acanthamoeba Keratitis. Invest. Ophthalmol. Vis. Sci. 2021;62(3):33. doi: https://doi.org/10.1167/iovs.62.3.33.

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Abstract

Purpose: Over a third of patients with Acanthamoeba keratitis (AK) experience severe inflammatory complications (SICs). This study aimed to determine if some contact lens (CL) wearers with AK were predisposed to SICs due to variations in key immune genes.

Methods: CL wearers with AK who attended Moorfields Eye Hospital were recruited prospectively between April 2013 and October 2014. SICs were defined as scleritis and/or stromal ring infiltrate. Genomic DNA was processed with an Illumina Low Input Custom Amplicon assay of 58 single nucleotide polymorphism (SNP) targets across 18 genes and tested for association in PLINK.

Results: Genomic DNA was obtained and analyzed for 105 cases of AK, 40 (38%) of whom experienced SICs. SNPs in the CXCL8 gene encoding IL-8 was significantly associated with protection from SICs (chr4: rs1126647, odds ratio [OR] = 0.3, P = 0.005, rs2227543, OR = 0.4, P = 0.007, and rs2227307, OR = 0.4, P = 0.02) after adjusting for age, sex, steroids prediagnosis, and herpes simplex keratitis (HSK) misdiagnosis. Two TLR-4 SNPs were associated with increased risk of SICs (chr9: rs4986791 and rs4986790, both OR = 6.9, P = 0.01). Th-17 associated SNPs (chr1: IL-23R rs11209026, chr2: IL-1β rs16944, and chr12: IL-22 rs1179251) were also associated with SICs.

Conclusions: The current study identifies biologically relevant genetic variants in patients with AK with SICs; IL-8 is associated with a strong neutrophil response in the cornea in AK, TLR-4 is important in early AK disease, and Th-17 genes are associated with adaptive immune responses to AK in animal models. Genetic screening of patients with AK to predict severity is viable and this would be expected to assist disease management.

Acanthamoeba keratitis (AK) results in the most prolonged and severe morbidity of any of the causes of acute microbial keratitis.1 Unlike other Acanthamoeba infections, which usually affect immunocompromised patients, AK occurs in immunocompetent healthy individuals.2 Treatment failure rates are currently in the order of 50% for a poor outcome (acuity less ≤ 20/80 ± surgery) with median overall cure times in the order of 5 months3,4 and 25% of patients requiring more than 9 months of treatment.3 Over 25% of patients in large series have required a keratoplasty.5,6 
One of the major causes of morbidity in AK is the severe inflammatory response that develops in some patients.7 This occurs in both the infected cornea, with the development of a corneal ring infiltrate, as well as in the usually noninfected adjacent sclera. Scleritis causes both severe pain, and, in some cases, the secondary complications of scleral thinning, choroidal detachment, and hypotony.811 Corneal ring infiltrates are reported in 13 to 24% of cases and scleritis in 9 to 18% of patients with AK.8,12,13 These severe inflammatory complications are associated with poor outcomes.7 
We and others have shown that single nucleotide polymorphisms (SNPs) of inflammatory genes involved in animal models of bacterial keratitis are associated with the onset and severity of corneal infection in patients who wear contact lenses (CLs).1418 CL wear is the major risk factor for AK in developed countries, accounting for around 90% of cases.19 This study was designed to test our hypothesis that differences in immune gene variation in otherwise healthy CL wearers determine whether a severe inflammatory response develops in the course of AK. 
Methods
This was a prospective study of patients with CL associated AK classified as either displaying a severe inflammatory complication (SICs) or not (non-SICs). 
Ethics
The protocol was approved by the London-Hampstead National Research Ethics Service (Reference 13/LO/0032) and Moorfields Eye Hospital Research Management Committee. The trial was adopted by the UK National Institute for Health Research, Clinical Research Network Portfolio. All participants gave written informed consent and all study procedures conformed to the tenets of the Declaration of Helsinki. 
Recruitment
CL wearers who attended Moorfields Eye Hospital for diagnosis and/or ongoing treatment for AK were recruited prospectively between April 2013 and October 2014. 
For the diagnosis of AK, one or more of the following criteria was satisfied: 
  • 1. A positive Acanthamoeba culture or histopathologic confirmation of trophozoites or cysts.
  • 2. Culture-negative cases that were positively identified as having Acanthamoeba cysts on confocal microscopy, together with a typical clinical course and response to anti-amoebic treatment (AAT).
  • 3. In the absence of criterion 1 or 2, patients with perineural corneal infiltrates or a typical clinical course with a response to AAT.
Genomic DNA was collected by a self-administered cheek swab (FLOQswabs; Copan Diagnostics, CA, USA) or a saliva kit (DNAGenotek, Ontario, Canada or DNAgard, Biomatricia, CA, USA). Age, sex, and ethnicity was collected via self-report. Clinical data were collected retrospectively from the patients’ with AK hospital records using the REDCap database platform (Vanderbilt University, Nashville, TN, USA). 
Disease stage at presentation of AK was divided into 3 categories: stage 1, corneal epitheliopathy only; stage 2, presence of one or more of corneal epithelial defects, perineural infiltrates or stromal infiltrates, in addition to stage 1 findings; and stage 3, presence of a corneal stromal ring infiltrate in addition to one or more features of stage 2 disease.20 
Severe Inflammatory Complications
SICs were defined as documentation of scleritis and/or stromal ring infiltrate in the patients’ hospital records in accordance with a previous study.7 
SNP Assay Design
Much of the understanding of the immune response to Acanthamoeba has come from studies in animal models. These studies indicate that innate cells and proteins, such as TLR-4, IL-8, and neutrophils21,22 are crucial to mount a response in early AK disease, and that Th17 cells are involved in limiting disease severity.23 We therefore designed a SNP assay targeting Th-17 pathway genes (IL17-A, IL17-F, IL17-R, IL-1β, TNFα, TGFβ, IFNγ, IL-6, IL-23R, IL-22, and IL-27) and CXCL8 (encodes for IL-8). We also included Th-1 and Th-2 interleukins genes found associated with bacterial keratitis (IL-12 and IL-10, respectively) and a novel cytokine (IL-18). Surface defense proteins defensin 1 and TLR-4 were further targeted, as expression of these proteins are upregulated in the presence of Acanthamoeba in both animal models and in vitro.24 As a SNP of TNF-related apoptosis-inducing ligand receptor (TRAIL-R1), a negative modulator of inflammation, has recently been associated with trachoma infection in humans25 and it was also included in the SNP panel. 
Following collection via a cheek swab or a saliva kit, genomic DNA was extracted and processed using the Illumina MiSeq platform. Detailed gDNA sample processing and bioinformatics methods are included in Supplementary File S1
SNPs and Phenotype Association Analysis
The PLINK (version 1.07) software was used for association analysis.26 SNPs with a minor allele frequency < 0.01 in the study cohort or not conforming to Hardy-Weinberg Equilibrium (P < 0.001) were removed. Remaining SNPs were assessed for association with SICs using logistic regression. Topical corticosteroid use prior to the initiation of AAT, bacterial keratitis treatment prior to AAT, herpes simplex virus (HSV) keratitis treatment prior to AAT, disease stage at presentation, and tertiary referral (i.e. referred from another hospital for tertiary management at Moorfields Eye Hospital) were tested for univariate associations and included in the multivariate model if significant (P < 0.2) along with sex and age as covariates. Analysis of linkage disequilibrium between multiple SNPs in the same gene was performed using HaploView (version 4).27 
Results
Genomic DNA was obtained and analyzed for 105 cases of AK, 40 (38%) of whom experienced SICs (Table 1). The average age at the time of presentation for all AK cases was 36.9 ± 15.5 years, and there was no difference in age between those with SICs compared to those without (38.3 ± 17.3 compared to 37.9 ± 16.2, P = 0.906). Likewise, there was no difference in sex between the two groups (women: 20/40, 50% vs. 35/65, 54%, P = 0.841). “Topical corticosteroid use” and “herpes simplex virus (HSV) keratitis treatment prior to the initiation AAT” was significant P < 0.2 and was included in the genotype model. “Disease stage at presentation” was more advanced in the SICs group (P < 0.001) so this variable was not included in the final model due to collinearity. There was no difference between “bacterial keratitis treatment prior to AAT” and “patients referred to tertiary care” for SICs versus non-SICs and so these characteristics were also not included in the multivariate model. 
Table 1.
 
Distribution of Sample, Presenting History, and Clinical Features of Patients With and Without Severe Inflammatory Complications
Table 1.
 
Distribution of Sample, Presenting History, and Clinical Features of Patients With and Without Severe Inflammatory Complications
Of the 72 targeted SNPs, 6 were not able to be sequenced with the assay (IL1B rs1143627; IL10 rs6703630; TLR4 rs12377632; IL18 rs5744292; IL22 rs2227483; and IFNγ rs2069707), 5 did not conform to Hardy-Weinberg Equilibrium (P > 0.001, IL12B rs17860508; TNFα rs1799964; IL22 rs1012356; IFNγ rs2069705; and TGFB1 rs1800470), and three had minor allele frequencies of < 0.01 (CXCL8/IL-8 rs2227532; TRAIL-R1 rs2230229; and IFNγ rs2069709) in this cohort. The remaining 58 SNPs were included in the analysis. Table 2 details the allele frequencies of the analyzed SNPs and adjusted multivariable analysis by allele. The adjusted analysis accounted for the a priori factors of age at diagnosis, sex, topical corticosteroid use prior to AAT, and HSV keratitis treatment prior to AAT. Significant associations (adjusted P < 0.05) after adjustment for covariates were found in SNPs in genes IL-23R, IL-1β, CXCL8, TLR-4, and IL-22
Table 2.
 
Allelic Frequencies of Targeted SNPs and Association Analysis in Patients With AK With Severe Inflammatory Complications (SICs) Compared to Those Without
Table 2.
 
Allelic Frequencies of Targeted SNPs and Association Analysis in Patients With AK With Severe Inflammatory Complications (SICs) Compared to Those Without
The highest significance was found for CXCL8 (encodes IL-8) and TLR-4 SNPs and because an association was found with more than one SNP for each of these genes, they were investigated further for inheritance and linkage disequilibrium (LD). 
Associated SNPs in CXCL8 (encodes IL-8) and TLR-4 demonstrated dominant inheritance models (Table 3). LD was performed in HaploView (version 4) for the targeted SNPs in CXCL8 (encodes IL-8) and TLR-4 (Fig.). All SNPs in CXCL8 (encodes IL-8) were in LD apart from rs2227549 (4:73741020). For TLR-4 only the 2 associated SNPs were in an LD block with low LD across the rest of the gene. 
Table 3.
 
Genotype Frequencies of CXCL8 (Encodes IL-8) and TLR-4 in Patients With Severe Inflammatory Complications (SICs) and Controls
Table 3.
 
Genotype Frequencies of CXCL8 (Encodes IL-8) and TLR-4 in Patients With Severe Inflammatory Complications (SICs) and Controls
Figure.
 
Linkage disequilibrium structure of selected SNPs in regions of interest in CXCL8 (encodes IL-8) (A) and TLR-4 (B). LD blocks were created with the default algorithm in HaploView program (version 4.1) that creates 95% confidence bounds on the normalized coefficient of linkage disequilibrium (D′) considered being in strong LD where 95% of the comparisons made are informative. The coloring of the boxes depends on two scores, the logarithm of the odds (LOD) and D′, high association (LOD > = 2 and D′ = 1, red), low association (LOD < 2 and D′ = 1, blue), and no association (LOD < 2 and D′ < 1, white). LOD score are shown within the box if D′ < 1.
Figure.
 
Linkage disequilibrium structure of selected SNPs in regions of interest in CXCL8 (encodes IL-8) (A) and TLR-4 (B). LD blocks were created with the default algorithm in HaploView program (version 4.1) that creates 95% confidence bounds on the normalized coefficient of linkage disequilibrium (D′) considered being in strong LD where 95% of the comparisons made are informative. The coloring of the boxes depends on two scores, the logarithm of the odds (LOD) and D′, high association (LOD > = 2 and D′ = 1, red), low association (LOD < 2 and D′ = 1, blue), and no association (LOD < 2 and D′ < 1, white). LOD score are shown within the box if D′ < 1.
Discussion
This study investigated SNPs in genes encoding innate and adaptive immune responses thought to play key roles in the severity of AK from in vitro and in vivo animal and human studies. SNPs in five genes encoding biologically relevant molecules, CXCL8 (encodes IL-8, rs2227543 C/T, rs1126647 A/T, and rs2227307 T/G, all in 3'UTR gene region) and TLR-4 (rs4986790 A/G, rs4986791 C/T, missense coding gene region), IL-1β (rs16944, G/A, upstream regulatory region), IL-23R (rs11209026, G/A, missense coding gene region), and IL-22(rs1179251, C/G, intron gene region) were identified that were associated with SICs in AK after adjusting for covariates. SNPs of CXCL8 (encoding IL-8) and TLR-4 had two significantly associated SNPs, each in LD and exhibited the strongest association with SICs. 
The CXCL8 (encodes IL-8) SNPs were protective for SICs in this population. Carriers of the minor allele (T) were around 70% less likely to experience SICs than noncarriers in this study. In this same AK patient cohort, increased levels of IL-8 protein in tears were associated with more severe AK disease.28 However, tear IL-8 levels with were not correlated with specific genotypes in patients in this study (P = 0.324). Similarly, a study of sepsis susceptibility in Chinese men29 found that the T allele for SNP rs1126647 was protective and increased levels of serum IL-8 were associated with sepsis in their population, but found no correlation between specific genotypes and the serum IL-8 protein levels. These findings indicate that this SNP may not be the causal SNP that increases the transcriptional capacity for IL-8. Other studies have suggested that rs4073 and rs2227306 may be causal SNPs.30 Although significance associations of these SNPs were not found in this study, they are in LD with those identified as significant. 
IL-8 is produced in the cornea by epithelial cells, keratocytes,31 and macrophages22 and has been shown to be upregulated in other ocular surface diseases, such as allergy32 and dry eye.33 IL-8 mobilizes and activates neutrophils, which is crucial in the early stage of corneal infection. However, activated neutrophils can also release metalloproteinases, which can degrade the extracellular matrix.34 This is evidenced by an immunodeficient mouse model, which when infected with adenovirus vector encoding human IL‐8, results in neutrophil infiltration and corneal ulceration.35 Thinning of the cornea and persistent epithelial defects, whereas not by definition part of SICs, are often present in severe cases of AK.19 IL-8 is produced early in the inflammatory cycle, but unlike most other inflammatory cytokines, it can remain active up to weeks at the site of inflammation.36,37 In contrast to the transitory effect of other proinflammatory cytokines that may last only a few hours, IL-8 may have a sustained influence36,37 and therefore could be an important contributor to severe inflammatory outcomes in AK. In addition, IL-8 may be repeatedly produced by epithelial and inflammatory cells, leading to prolonged inflammation and collateral tissue damage. 
Although IL-8 may have detrimental proinflammatory effects mediated through neutrophils, several animal studies have shown the powerful anti-acanthamoeba action of neutrophils in the early stages of disease. Neutrophils are chemotactic to Acanthamoeba trophozoites and cyst lysates, and are capable of killing the organism in vitro through myeloperoxidase (MPO).38 Upon injection of one million Acanthamoeba castellani trophozoites into the anterior chamber of a mini pig model, AK was eliminated by a robust neutrophil response.39 Intraocular infection has only been reported in a handful of human cases8 indicating this neutrophil response in the anterior segment may be a factor in limiting the organism from infecting the posterior eye in humans. 
In contrast to CXCL8, minor alleles of TLR-4 SNPs were associated with more severe AK in this study. Carriers of the minor allele were around 6.9 times more likely to experience SICs than noncarriers. The two significant TLR-4 SNPs are in almost complete LD. These SNPs are not common, however, the non-SIC minor allele frequencies are similar to population controls (minor allele frequency 0.06 from phase III 1000 Genomes) with increased frequency in SICs, reflecting a representative sample. 
TLR-4 is a surface recognition receptor present on corneal and conjunctival cells. In the presence of Acanthamoeba, in vivo and in vitro models both show activation of TLR-4 receptor.40 In mouse models of AK, TLR-4 is also involved in initiating the cytokine complex.40 It is likely that in patients with AK, decreased recognition of Acanthamoeba allows a greater inoculum load into the cornea with dysregulation of the cytokine response, increasing disease severity. TLR-4 is primarily associated with a pro-inflammatory response, however, chronic activation of TLR-4 can lead to an anti-inflammatory response in some circumstances.41 If this is the case in AK, patients with milder disease may be able to mount this anti-inflammatory response, whereas those with the SNP and more severe disease may not, leading to chronic hyperinflammation. 
A study of patients with bacterial keratitis in India found higher carriage of TLR-4 rs4986791 SNP compared with healthy controls.18 However, another study did not find an association in rs4986790 (in LD with rs4986791) between CL patients with bacterial keratitis compared with healthy CL wearing controls in a Caucasian population.14 The differences in results in these two studies are intriguing, but may be attributable to the rarity of the minor alleles. Interestingly, carriers of TLR-4 SNPs have been shown to have a decreased response to endotoxins in several studies.42,43 Of note, patients with TLR-4 SNPs show a blunted airway response to inhalation of lipopolysaccharide (LPS).42 The minor (T) allele of rs4986791 is associated with lower TLR-4 expression in blood plasma of a large German cohort, which is replicated in a Arab and Asian cohort of healthy individuals.44 
Three Th17-related genes were associated with SICs in this study, IL-1β, IL-23R, and IL-22. IL-1β and IL-22 are proinflammatory cytokines and associated with the differentiation of CD4+ T cells into effector Th17 cells. For IL-22, the SNP (rs1179251), as for CXCL8 (encodes IL-8) was protective. IL-22 tear fluid protein was also expressed more often in this cohort of patients with AK who experienced severe disease, as for IL-8.28 However, we did not find an association between IL-22 tear protein levels and rs1179251 genotypes (P = 0.07). To our knowledge, there are no studies that correlate IL-22 protein to rs1179251. Of note is that serum IL-22 protein is increased in idiopathic scleritis.45 A study of IL-22 gene variations in autoimmune patients found the CC wild type genotype was associated with Graves’ ophthalmopathy, conferring protection by the minor allele, similar to our findings.46 Serum levels of IL-22 are associated with the minor allele of rs2227484,47 and stimulated peripheral blood mononuclear cells (PMBC) from patients carrying the minor allele of rs2227473 produce more IL-22.48 However, both these SNPs are in the promotor region of the gene, whereas rs1179251 is in intron 4, rendering it difficult to draw comparisons. 
In alignment with other cytokines in this study, the significant detection of SNP in IL-1β (rs16944) is associated with a lower risk of SICs. This SNP minor allele is associated with increased gene expression in PBMCs.49 However. IL-1β tear protein was not detectable in any of the tear samples from AK cases or nonaffected healthy controls in this current study population.28 This SNP and others in IL-1β have been associated with susceptibility to early onset periodontitis50 and osteoarthritis.51 In a previous study in a Caucasian population, an association was not found with IL-1β SNPs between patients with CL associated bacterial keratitis and healthy CL wearing controls.14 Further work is required to understand the role of IL-1β in AK. 
For IL-23R rs11209026, carriage of the A allele was associated with a 6 times increased risk of SICs. The presence of the A allele increases expression of the soluble form of IL-23R mRNA (which then functions as a decoy receptor) and thus impairs the function of IL-23 in its ability to promote and maintain Th-17 cells.52 This may mean that the Th-17 responses observed in an animal model of AK23 are hampered in patients with this SNP. 
There are several limitations of this study, in particular the sample size. Although this is a rare disease and we have a relatively large sample size per se, this limits power. In addition, the small sample size did not enable a more comprehensive analysis, such as genomewide association study (GWAS) or exome screening. Similarly, we have not made a correction for multiple testing of the 58 SNPs. All of the SNPs analyzed in this study had a priori hypothesis of association based on previous literature, but none reach significance under strict multiple testing criteria, despite the large effect sizes in many instances. Replication of these findings in similar, but independent, cohorts is critical and meta-analyses will likely be required to reach true statistical significance. Another limitation in this rare cohort was the self-report of ethnicity, which was completed poorly by participants (76/105, 72% identified ethnicity), however, 70/76, 92% identified as Caucasian. In a previous genetic study of bacterial keratitis in CL wearers at Moorfields Eye Hospital in 2012, 82% were Caucasian,14 adding certainty that the cohort in this current study is predominantly Caucasian. 
Nevertheless, the study author's clinical experience in this area meant a very well phenotyped cohort. In addition, a well-matched cohort in terms of sex and age between the SICs and non-SIC groups were recruited. Our results are further strengthened by an earlier published tear fluid protein study carried out on the same patient cohort.28 
In summary, our study has highlighted the importance of innate and Th17 immunity in patients with AK. A study of gene expression in late stage bacterial and fungal keratitis also found persistence of innate immune pathways.53 Further work will be important to define cause and effect in tissue and animal models. 
Furthermore, this study demonstrates that genetic screening of patients with AK for the presence of these SNPs is viable and could predict the risk of patients developing the severe inflammatory complications of the disease. The presence of these SNPs in an individual patient could assist clinicians managing AK by identifying patients susceptible to developing SICs in whom the prompt treatment of inflammation can be expected to improve outcomes. 
Acknowledgments
The authors thank Scott Hau, Melanie Mason, and the Cornea and External Disease Department at Moorfields Eye Hospital for assistance with recruitment. The authors also extend thanks to the Barts and London Genome Facility at Queen Mary, London University, for laboratory processing. 
Financial support was received from Fight for Sight (1465/6), Moorfields Eye Hospital Special Trustees (ST 12 09A), and anonymous donors to Moorfields Eye Charities. None of the funders had a role in the design or conduct of this research. Carnt was supported by an NHMRC Early Career CJ Martin Fellowship (APP1036728). Part of both Carnt and Dart's salary was paid by the National Institute of Research (NIHR) Biomedical Research Centre (BRC) at Moorfields Eye Hospital NIHR BRC. The study was adopted under the (NIHR) Clinical Research Network (CRN) Portfolio (Ref 14554) which provided infrastructure support. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health. 
Disclosure: N.A. Carnt, None; I. Pang, None; K.P. Burdon, None; V. Calder, None; J.K. Dart, None; D.Subedi, None; A.J. Hardcastle, None 
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Figure.
 
Linkage disequilibrium structure of selected SNPs in regions of interest in CXCL8 (encodes IL-8) (A) and TLR-4 (B). LD blocks were created with the default algorithm in HaploView program (version 4.1) that creates 95% confidence bounds on the normalized coefficient of linkage disequilibrium (D′) considered being in strong LD where 95% of the comparisons made are informative. The coloring of the boxes depends on two scores, the logarithm of the odds (LOD) and D′, high association (LOD > = 2 and D′ = 1, red), low association (LOD < 2 and D′ = 1, blue), and no association (LOD < 2 and D′ < 1, white). LOD score are shown within the box if D′ < 1.
Figure.
 
Linkage disequilibrium structure of selected SNPs in regions of interest in CXCL8 (encodes IL-8) (A) and TLR-4 (B). LD blocks were created with the default algorithm in HaploView program (version 4.1) that creates 95% confidence bounds on the normalized coefficient of linkage disequilibrium (D′) considered being in strong LD where 95% of the comparisons made are informative. The coloring of the boxes depends on two scores, the logarithm of the odds (LOD) and D′, high association (LOD > = 2 and D′ = 1, red), low association (LOD < 2 and D′ = 1, blue), and no association (LOD < 2 and D′ < 1, white). LOD score are shown within the box if D′ < 1.
Table 1.
 
Distribution of Sample, Presenting History, and Clinical Features of Patients With and Without Severe Inflammatory Complications
Table 1.
 
Distribution of Sample, Presenting History, and Clinical Features of Patients With and Without Severe Inflammatory Complications
Table 2.
 
Allelic Frequencies of Targeted SNPs and Association Analysis in Patients With AK With Severe Inflammatory Complications (SICs) Compared to Those Without
Table 2.
 
Allelic Frequencies of Targeted SNPs and Association Analysis in Patients With AK With Severe Inflammatory Complications (SICs) Compared to Those Without
Table 3.
 
Genotype Frequencies of CXCL8 (Encodes IL-8) and TLR-4 in Patients With Severe Inflammatory Complications (SICs) and Controls
Table 3.
 
Genotype Frequencies of CXCL8 (Encodes IL-8) and TLR-4 in Patients With Severe Inflammatory Complications (SICs) and Controls
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