A Scan of Pleiotropic Immune Mediated Disease Genes Identifies Novel Determinants of Baseline FVIII Inhibitor Status in Hemophilia-A

Abstract Hemophilia-A (HA) is caused by heterogeneous loss-of-function factor (F)VIII gene ( F8 )-mutations and deficiencies in plasma-FVIII-activity that impair intrinsic-pathway-mediated coagulation-amplification. The standard-of-care for severe-HA-patients is regular infusions of therapeutic-FVIII-proteins (tFVIIIs) but ~30% develop neutralizing-tFVIII-antibodies called “FVIII-inhibitors (FEIs)” and become refractory. We used the PATH study and ImmunoChip to scan immune-mediated-disease (IMD)-genes for novel and/or replicated genomic-sequence-variations associated with baseline-FEI-status while accounting for non-independence of data due to genetic-relatedness and F8 -mutational-heterogeneity. The baseline-FEI-status of 450 North American PATH subjects—206 with black-African-ancestry and 244 with white-European-ancestry—was the dependent variable. The F8 -mutation-data and a genetic-relatedness matrix were incorporated into a binary linear-mixed model of genetic association with baseline-FEI-status. We adopted a gene-centric-association-strategy to scan, as candidates, pleiotropic-IMD-genes implicated in the development of either ³2 autoimmune-/autoinflammatory-disorders (AADs) or ³1 AAD and FEIs. Baseline-FEI-status was significantly associated with SNPs assigned to NOS2A (rs117382854; p=3.2E-6) and B3GNT2 (rs10176009; p=5.1E-6), which have functions in anti-microbial-/-tumoral-immunity. Among IMD-genes implicated in FEI-risk previously, we identified strong associations with CTLA4 assigned SNPs (p=2.2E-5). The F8 -mutation-effect underlies ~15% of the total heritability for baseline-FEI-status. Additive genetic heritability and SNPs in IMD-genes account for >50% of the patient-specific variability in baseline-FEI-status. Race is a significant determinant independent of F8 ‑mutation-effects and non- F8 -genetics.


INTRODUCTION
Hemophilia-A (HA) is caused by X-linked factorVIII (FVIII) gene (F8) mutations and variable de ciencies in plasma FVIII coagulant activity (FVIII:C).Bleeding propensities in HA patients are inversely correlated with their baseline (i.e., untreated) FVIII:C levels and are classi ed as mild (5%£FVIII:C<40%), moderate (1%£FVIII:C<5%), or severe (FVIII:C<1%). 1,2Because recurrent hemarthrosis causes crippling joint disease, prophylactic infusions with plasma-derived (pd)-or recombinant (r)-therapeutic-FVIIIproteins (tFVIIIs)-called "products" herein-begin for severe HA patients when toddlers.However, ~30% of severe HA patients develop neutralizing anti-tFVIII-antibodies called "FVIII-inhibitors (FEIs)" which leave them refractory to these products and signi cantly increase morbidity and mortality. 3,4e immunogenicity of tFVIIIs is a complex trait with both environmental and genetic determinants deriving from several variable immunologically relevant characteristics of different products, treatment regimens, and patients. 5,6,7,8,9,10The highly heterogeneous causative-F8-mutations identi ed in unrelated HA patients has repeatedly been shown to comprise a potent genetic determinant of FEI risk. 5,6In a systematic review of data from >5,000 severe HA patients, Gouw et al. conducted a meta-analysis to estimate the relative risk of FEI development conferred by different categories of F8 mutation types. 11In that report, the group of patients with any large deletion involving ³2 exons had an ~3-4x greater risk of FEI development than those with either distal or proximal intron (I) 22-inversions (invs), which account for ~45% of all newborns with severe HA, while the patients with I22-invs had an ~3x greater risk of FEIs than the patients with any missense single-base-substitution-mutation (SBSM), which is consistent with a contemporaneous report. 12Another recent study-conducted on a group of >1,200 non-severe HA patients with F8 missense-SBSMs causing either moderate HA (~15%) or mild HA (~85%)-found comparable heterogeneity in FEI risk among subjects predicted to express endogenously almost 200 distinct mutant-FVIII-proteins, with most likely differing at only single amino acid residues. 13aken together, these ndings highlight the substantial F8 mutation heterogeneity in relation to FEI risk, which we account for-for the rst time-in this investigation.
An issue that needs to be addressed in genetic analysis of FEI risk is the widely observed fact that study samples tend to be composed of a sizeable fraction of related individuals.In certain studies, like the Malmö International Brother Study, a large fraction of genetic relatedness arises literally by design. 14n other studies, such as the current study, a lower level of genetic relatedness may arise in the sample because close relatives (e.g., brothers, male cousins, uncle-nephew pairs, and grandfather-grandson pairs) and even more distant relatives often receive medical care at the same specialized regional hemophilia treatment center (HTC).For example, in an investigation by Viel et al., for which HA patients were recruited from ve HTCs in the southeastern US, 31% of the subjects were closely related to at least one other subject as recorded in their study questionnaires by the enrolling nurses. 15Thus, HTC-based recruitment appears more likely to be enriched for relatives as compared to random sampling of a population; importantly, this leads to the statistical problem of non-independence due to genetic relatedness.
The statistical methods we developed, and advocate herein, control for both F8 mutation heterogeneity and genetic non-independence and are a straightforward extension of the linear mixed model analytic approaches that our group has been developing to study the genetics of complex diseases/traits. 16,17,18,19Speci cally, we applied these methods to data from the Personalized Alternative Therapies for Hemophilia (PATH) study, which was designed to replicate and mechanistically clarify non-HA variants in F8 found previously to be associated with the greater frequency of FEIs observed for HA patients self-reporting black-African (BA)-compared to white-European (WE)-racial/ethnic-ancestry. 15he results from several association studies of different candidate genes for autoimmune-/autoin ammatory-disorders (AADs)-referred to as immune-mediated diseases (IMDs) as they are caused by disorders of the adaptive and innate components of the immune system, respectively-have together identi ed variants in 25 such loci, which may contribute to the variable frequency of pathologic immunogenicity observed for the tFVIIIs infused in different HA patients. 20,21,22,23,24,25,26,27,28,29,30,31These results were often con icting, however, and none have undergone true independent con rmation with functional validation.We used PATH genotyping datafrom the single-nucleotide-polymorphisms (SNPs) and -variants (SNVs) assigned to the candidate ImmunoChip (IC) genes that have previously been implicated in the susceptibility to developing FEIs and/or AADs-in a gene-centric association-scan (GCAS) to identify novel and/or replicated IMDgenes that in uence baseline-FEI-status. 32,33 MATERIAL AND METHODS Patients, questionnaires, and blood samples Between January 2010 and December 2011, we invited North American HA patients managed clinically at any of 20 participating regional HTCs (14 in the US and six in Canada) to enroll in the IRB-approved PATH study based at the Los Angeles Veterans Affairs Medical Center (CA, USA) (IRB: 2009-091280) and Bloodworks Northwest (WA, USA) (IRB: 13018). 34For details on the PATH study's cohort, inclusion and exclusion criteria, data collection instrument, blood samples obtained, and assays performed (see Supplemental Appendix).
Bethesda assays for detecting and quantifying FEIs, determining historical-and baseline-FEI-status, and assessing FEI risk As described by Pandey et al., 34 we determined each PATH subject's: 1) historical-FEI-status by considering the results from all Bethesda assays 35,36,37 performed before enrollment-during regularly scheduled HTC visits-which were obtained via medical chart review and provided to us in their questionnaires; and 2) baseline-FEI-status based on results from the single Bethesda assay performed on study entry.See Supplemental Appendix for details on the Bethesda assays performed, including the cutoffs used to interpret the results with respect to the presence or absence of FEIs, and assessment of FEI risk.
Identi cation of HA-causing F8-mutations and creation/use of the shared F8-mutation-matrix As described previously Pandey et al., 34 the F8 mutations in HA patients were identi ed with three distinct F8-speci c assays that involved Sanger resequencing, RT-PCR, and PCR, 38 which were also employed in the Pharmacogenetics of Inhibitor Risk study by Viel et al., except that a long-range PCR (instead of an RT-PCR) assay was employed to detect I22-invs. 15See Supplemental Appendix for details on the three assays performed, including the speci c F8 mutation type(s) identi ed (or not identi ed) by each and their QC criteria, as well as the design/creation and use of the F8-mutation-matrix.

Preparation and quality control of IC genotyping data
Genomic-DNA from each PATH subject was subjected to genotyping at the nearly 200,000 sequence variations that can be interrogated simultaneously on the IC (Illumina; San Diego, CA, USA). 32,33,39See Supplemental Appendix for details on the genotyping assay including the: IC version used; types and chromosomal locations of the variations genotyped; QC criteria for variations yielding high-quality genotypes; and the numbers of genotyped variations used in the (i) empiric-kinship/genetic-relationship matrix and (ii) GCAS of candidate pleiotropic-IMD-genes. 40-based empiric-kinship/genetic-relationship matrix All IC-variations with high-quality genotypes (n=137,776) were used to compute the pairwise genetic correlation between the PATH study subjects and to create the empiric kinship/genetic relationship matrix.See Supplemental Appendix for details on creating and utilizing the empiric kinship/genetic relationship matrix.

Novel gene-centric association-scan (GCAS) of pleiotropic-IMD-genes
Because results from prior studies of immune-response-genes/-gene-variations have been con icting and none have been con rmed with functional validation, we employed new statistical methods-that, as described under the next sub-header, controlled for F8 mutation heterogeneity and genetic nonindependence-in a GCAS focused on pleiotropic-IMD-genes, which met the extra criterion for candidates previously implicated in the development of FEIs and/or AADs. 20,21,22,23,24,25,26,27,28,29,30,31,32,33,See Supplemental Appendix for more details.
Linear-mixed model with an additional F8-mutation-type component to evaluate risk of being FEI + on study entry We analyzed baseline-FEI-status (Yes versus No) for associations with the subsets of candidate ICsequence-variations under a binary linear mixed model 16,17 given as follows: where for the -th individual denotes the baseline-FEI-risk value, denotes the vector of covariates, is the intercept parameter, and is the vector of regression coe cients.See Supplemental Appendix for more details on how we: 1) selected/examined covariates; 2) accounted for potential sources of nonindependence; 3) derived odds ratios (ORs); 4) calculated/used heritabilities; and 5) analyzed the genetic effect of each high-quality SNP and SNV-comprising the subsets of variations which were assigned by consensus of the IC-consortium to one of the candidate pleiotropic-IMD-genes known or suspected to in uence the development of ³2 distinct AADs (n=125) and/or FEIs (n=22)-for its contribution to baseline-FEI-status by means of a likelihood-ratio-test as implemented in SOLAR.
Because the baseline-FEI-status of HA patients has never been investigated genetically previously, we estimated its heritability for the rst time herein.(Though the genetics associated with FEI risk has been reported on extensively previously, the dependent variable in prior studies has been "lifetime-FEI-status", i.e., what we refer to here as historical-FEI-status, which is: 'YES', if a patient has ever developed FEIs, of AT, at any time in life, irrespective of whether or not a FEI is present at 'baseline', on study entry; or 'NO', if a patient has never developed FEIs, of AT, at any time in life, including at 'baseline', on study entry.)We found that the residual additive genetic heritability for the presence of FEIs of AT (FEI AT + ) on entry into the PATH study versus their absence (FEI AT -) was 0.47 (p<0.05) and the F8-mutation-speci c heritability was 0.08 (p<0.05), the latter of which comprised ~15% of the total observable heritability (0.08/0.08+0.47).
We leveraged the substantial signi cant additive genetic and F8-mutation-speci c heritabilities for baseline-FEI-status to perform a GCAS in which the 101 distinct pleiotropic-IMD-genes on the IC-that met the criterion of having previously been implicated in the risk of developing either two or more distinct AADs (n=76) or FEIs and at least one AAD (n=21), or both (n=4)-were screened as candidate determinants for the presence or absence of FEIs (on study entry) in these previously-treated-patients (PTPs) with HA.
This pleiotropic-IMD-gene is expressed by regulatory T-cells and encodes IL-10, an anti-in ammatory cytokine that helps balance immune responses, allowing clearance of infectious microbes but minimizing host damage. 64The IL10-superfamily was identi ed via rs7528265 (206,845,818 C>T)-an IL10assigned SNP located ~73kbp upstream from its initiation-codon-which revealed a suggestive genotypespeci c increase in FEI risk (p=3.7E-3)(not shown).

Multivariate logistic regression odds ratios
Having identi ed a novel role for NOS2A and B3GNT2¾and veri ed the previously implicated role for CTLA4 and IL10¾in FEI risk, we incorporated the top SNP assigned to each of these pleiotropic-IMDgenes altogether in a model, along with race, to evaluate their joint ability to predict baseline-FEI-status where all effects are additive.The OR and 95%-con dence interval (CI) lower-and upper-bound (LB and UB)¾reported as OR (95% CI LB, 95% CI UB)¾for rs117382854, rs10176009, rs231780, rs7528265, and race, respectively, were  4A, covariates A-E), which indicate that the MAs of three of these four IMD-genevariations (i.e., NOS2, B3GNT2, and IL10) increased the risk of having FEIs of AT on study entry while the true MA of CTLA4 and WE-racial/ethnic-ancestry were protective.
Because the main goal of the PATH study was to con rm and further clarify the in uence of race on FEI development, we sought to explicitly evaluate whether its role is mediated in part via genetic effects by Bayesian model selection of all possible interactions of race with these four pleiotropic-IMD-genevariations.65 The best model identi ed involved a single interaction term between race and B3GNT2 (i.e., rs10176009), where rs117382854, rs10176009, and race, by itself, still exerted signi cant additive effects (Figure 4B, covariates F-J).The ORs and 95% CIs for rs117382854, rs231780, rs7528265, race×rs10176009, and race alone are respectively 3.00 (1.

DISCUSSION
Prior studies aimed at identifying inherited determinants of FEI development in PTPs have exclusively examined historical-FEI-status and¾except for a few which recently employed genome-wide scans 66,67,68 ¾have used candidate-gene-based approaches that focused on sequence variation in or near either F8 (i.e., mutation typesand/or non-HA-causing ns-SNPs) 5,6,7,8,9,10 or essential immune system genes. 20,21,22,23,24,25,26,27,28,29,30,31While ndings from these studies have clearly demonstrated the importance of F8 mutation type, they have been con icting with respect to the role played by F8 ns-SNPs and IMD-genes/-gene-variants. 9,20,21,22,23,24,25,26,27,28,29,30,31,34,44,69,70,71,72 Speci c mechanisms underlying the differential effects of some F8 mutation types are being elucidated and appear to variably involve interrelated processes regulating immunologic-tolerance (IT) and -reactivity (IR). 8,11,13,34,43,44In this context, IT refers to whether a given patient's abnormal F8 encodes a polypeptide(s)¾if indeed one or more is encoded¾that, except for the residue(s) resulting from the speci c mutation(s), contain the entire amino acid sequence of his tFVIII, i.e., the endogenous material needed to induce IT to his "self" FVIII, which theoretically would provide preexisting-immunologic-unresponsiveness to the identical portions of his tFVIII.The process of IR relates to whether one of the limited number of distinct allotypes in the repertoire of HLA-class-II (HLAII) molecules expressed by dendritic cells and B-cells in a given HA patient (i.e., which ranges from 3-12) is able to present-as a neoepitope to his CD4 T-cells, threshold levels of at least one "foreign" peptide liberated from his tFVIII via intracellular proteolytic processing. 73,74We presented ndings from the rst study of baseline-FEI-status, a novel outcome for PTPs that appears to be affected distinctly by what we have coined the "immunogenicity balance"-a complex balance of processes relating to IT and IR-than historical-FEI-status as the presence of FEIs on study entry likely also interrogates determinants in uencing whether an immune response is persistent or resolves in addition to its induction/development.

Accounting for F8 mutational heterogeneity and genetic relatedness
We identi ed causative-F8-mutations in 406 of the 450 HA patients enrolled in PATH (90.2%), including 188 of the 206 with BA (91.3%) versus 218 of the 244 with WE (89.3%) racial/ethnic-ancestry.Although F8 abnormalities were not found in 44 patients (9.8%)-for the various reasons described above, which were likely variably at play in prior studies as causative-F8-mutations are frequently not identi ed in between 7.5-15% of subjects-these were comparably disbursed across the groups self-reporting BAversus WE-ancestry, i.e., 8.7% versus 10.7%, respectively.The F8 abnormalities identi ed in PATH were highly heterogeneous-comprising 132 distinct loss-of-function mutations-across both groups of subjects.This included the highly-recurrent bi-allelic I22-invs and the allelically highly-diverse missense-SBSMs-the two most common causative-F8-mutation types-which, among the US subjects respectively accounted for 31.9% and 32.4% versus 42.7% and 19.0% of those with BA-versus WE-racial/ethnicancestry, respectively.This extreme heterogeneity among causative-F8-mutations has been found in all racial and ethnic groups of patients studied thus far. 15,67,68,75,76To account for mutational heterogeneity appropriately/accurately, we also quanti ed the number of causative-F8-abnormalities in each of the other categories identi ed.Because this heterogeneity has not been accounted for rigorously in prior studies of FEIs, we achieved this herein by employing a shared causal-F8-mutation matrix to estimate the mutational effect.Our linear mixed model can be conceptualized in terms of the: 1) signal-to-noise ratio, where statistical noise arises from both the (i) non-independence that derives from genetic relatedness in the samples, and (ii) large degree of causative-F8-mutational heterogeneity (and is accounted for by the random effects part of the model); and 2) main genetic signals of interest that result from the SNPs/SNVs (and is accounted for by the xed effects part of the model).
In association studies with a non-negligible number of related patients (e.g., full-and half-sibs, rstcousins, and avuncular-relations), it is important to account for the genetic relatedness of the subjects.However, the extensive amount of genetic relatedness existing in many prior studies of FEIs has not been adequately accounted for.Two types of genetic relatedness exist from familial relationships among subjects including those known and those unknown but obtained from empirical estimates of relatedness over a panel of genetic markers.Our empirical kinship matrix estimates both types, as they belong to the same relatedness continuum (see Supplementary Figure 1).The extent of relatedness in such studies results in a tacit violation of the independence-of-data assumption.Therefore, modeling this nonindependence is a crucial feature of our approach.It is important to emphasize that accounting for the non-independence due to both relatedness and causative-F8-mutation heterogeneity helps to optimize the e ciency of parameter estimation in the xed effects.

Fixed effects modeling of baseline-FEI-status
Novel IMD-genes/-gene-variants involved in FEI risk Using our GCAS approach and the high-quality genotyped IC-SNPs/-SNVs assigned to the 101 candidategenes interrogated, we discovered a novel role for NOS2A and B3GNT2-two pleiotropic-IMD-genes known to in uence the development of ³2 AADs but not FEIs-as determinants of baseline-FEI-status.We also discovered that CTLA4 and IL10-two additional pleiotropic-IMD-genes known to in uence the development of (i) ³2 AADs, and, in contrast to NOS2A and B3GNT2, (ii) FEIs, i.e., historical-FEI-status-are determinants of baseline-FEI-status via respectively four SNPs in CTLA4 and one novel SNP in the IL10superfamily, despite the fact that both genes have previously been implicated and (now) independently con rmed to play a role in FEI risk.The novel SNP of CTLA4, which is in I3, remained signi cantly associated after correction for multiple testing (data not shown).The novel SNP of IL10, which is in the upstream regulatory sequence, was marginally associated with FEIs but became only suggestively associated after multiple testing correction (data not shown).

Role of race in FEI risk
Under the additive model, race had an independent effect on FEI risk.Speci cally, we observed that HA patients self-reporting BA-racial-ancestry had a higher prevalence of FEIs (i) historically (35.1%) compared to the patients with WE-racial-ancestry (26.6%), and (ii) at study entry (21.5%) compared to the WE-racial-ancestry patients (15.9%), which is consistent with what is commonly reported (although marginally not signi cant: p=0.06). 10,14,15,71,72,77,78,79Of note, past studies that identi ed self-reported BA-racial-ancestry to be a determinant of risk for FEIs, utilized historical-FEI-status only, not baseline-FEIstatus.
Under the interaction model, we identi ed an interaction between race and rs10176009 (i.e., 62,282,826 C>A), a B3GNT2-assigned SNP located in the anking 3'-gDNA of this pleiotropic-IMD-gene, which is known to in uence susceptibility to at least ve distinct AADs.This interaction may be interpreted as suggesting that the MA of this SNP ('A') increases the risk of having any titer FEI (FEI AT + ) at baseline in HA patients but only in patients with BA-racial-ancestry as it is rare in those with WE-racial-ancestry.

Potential role of rare genetic variants in FEI risk
Because the IC was primarily constituted of IMD-gene-variations found in GWASs to in uence susceptibility to ³1 AADs, 32,33 and the genotyping-platforms used in these GWASs were comprised predominantly of by common (MA-frequencies [MAFs] >5%) and less common (1%£ MAFs £5%) SNPs, the majority of variations analyzed herein represent such SNPs (Supplementary Figure 2).Thus, rare SNVs (MAFs <1%) are infrequent on the IC.Moreover, as we have accounted for most of the F8-geneticeffects (i.e., mutation effect), the 47% additive genetic heritability may therefore be largely the result of rare variants.This is consistent with the important role that rare variants are now widely recognized as playing in complex traits. 80Notably, while MAs of the rst ve SNPs in Table 2-which were assigned to three pleiotropic-IMD-genes (NOS2A, B3GNT2, and CTLA4) and found in our GCAS to have the strongest associations with baseline-FEI-status-are of the less common or common types in patients with one racial-ancestry (i.e., two NOS2A-assigned SNPs in WE subjects; two B3GNT2-assigned SNPs and one CTLA4-assigned SNP in BA subjects), they are rare SNVs in subjects of the other racial-ancestry (i.e., two NOS2A-assigned SNVs in BA subjects; two B3GNT2-assigned SNVs and one CTLA4-assigned SNP in WE subjects).

Limitations
A limitation of PATH is under representation by mild and moderate HA patients in the cohort.Typically, up to 40% of subjects have non-severe HA when enrollment of patients-in developed countries, such as, e.g., in the US and Canada, which participated herein-occurs (i) during their scheduled bi-annual HTC appointments, and (ii) cross-sectionally, independent of their baseline-HA-severity and historical-FEIstatus.
As this is a study almost exclusively of PTPs, with most being older than 18 years of age, the tFVIII intravenous-infusion histories are invariably inaccurate with respect to important parameters such as the number of exposure days of a given tFVIII administered previously and the age at rst infusion of the initial tFVIII.Moreover, since most of the patients (almost 90%) have severe HA, the vast majority will have been on multiple different tFVIII products, including both pdFVIII and rFVIII, and within both of these broad categories of tFVIIIs, they likely will have received different brands (i.e., for pdFVIII products both those with and without pdVWF, and for rFVIII products, both those that are native full-length and those that are non-native with engineered B-domain deletions).

Conclusions
For the rst time in studies of FEIs in HA patients, we employed a statistical method that appropriately accounted for the non-independence of data due to both the sizeable genetic relatedness and F8 mutational heterogeneity which exists when subjects with Mendelian loss-of-function disorders are sampled.By employing this method in PATH, on IC-genotypes of variations (i.e., mainly SNPs but some SNVs with rare MAs and/or racially-restricted MAs) assigned to pleiotropic-IMD-loci known to in uence risk of developing two or more AADs, we identi ed: (i) the novel contribution of two genes (NOS2A and B3GNT2) to the risk of HA patients having FEIs on entering the study; (ii) additional genetic determinants of baseline-FEI-status in two pleiotropic-IMD-genes (CTLA4 and IL10 or, more accurately, the IL10-genesuperfamily) previously known to in uence FEI risk (i.e., historical-FEI-status); and (iii) an interaction between race and a B3GNT2-assigned SNP that may contribute to the greater risk of FEI development long known to occur in HA patients with self-reported BA-racial-ancestry.Finally, we established that a relatively sizeable genetic component of baseline-FEI-status remains to be identi ed and is likely due to gene-based variants with rare MAs.As such, identi cation of these currently unknown rare genetic variants will require larger studies with more related HA patients and either whole-exome-or wholegenome-sequencing to identify (and genotype) the rare variants not included on GWAS-chips.Manhattan plot of association results from an ImmunoChip (IC)-wide scan of baseline-FEI-status, highlighting the 10 SNPs assigned to the three pleiotropic-IMD-genes-including B3GNT2 on Chr2 (three solid green circles), CTLA4 on Chr2 (four solid orange circles), and NOS2A on Chr17 (three solid purple circles)-found to be signi cantly associated in the gene-centric scans shown at higher resolution in Figure 3.Each light-or dark-blue lled circle indicates one of the subset of 137,766 IC SNPs that yielded high-quality genotypes (i.e., passed QC as detailed in the Supplemental Appendix) but were not signi cantly associated with baseline-FEI-status at a signi cance threshold Bonferroni corrected for the multiple testing of the IC-wide set of 137,776 total SNPs that passed QC, i.e., they are all below the solid horizontal red line shown at the -log 10 (p-value) of 6.44, which would be the corrected threshold accounting for all successfully genotyped ImmunoChip SNPs.(Note that Bonferroni correction is de ned as the nominal signi cance threshold, i.e., a p-value < 0.05, divided by the total number of genotyped SNPs analyzed, which, in this case, was 137,776.)All 137,776 genotyped SNPs were used to create the empiric-kinship matrix that was utilized to account for the non-independence of data (due to both the known and unknown relationships among the study subjects) and estimate the heritability of baseline-FEI-status.The 10 SNPs signi cantly associated with baseline-FEI-status in the gene-centric scans included three, four, and three assigned respectively to B3GNT2, CTLA4, and NOS2A which fell above their respective -log 10 (p-value) thresholds of 3.75, 3.86, and 3.76 that are represented by the three horizontal red dashed lines (see Figure 3 for more details).

Figures Figure 1
Figures

Table 2
Because NO signals by activating soluble guanylyl cyclase, which in turn increases synthesis of cGMP from cellular GTP, NOS2 is a vital regulator of several physiologic processes especially those in the immune system related to host defense as NO has potent microbicidaland tumoricidal-actions. 46,47 Though NOS2A had not previously been implicated in the development of FEIs, nor in their persistence and/or resolution, it likely in uences the development of at least ve AADs, including multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid-arthritis (RA), ). Located on the proximal long-arm of chromosome (Chr)17, NOS2A comprises a centromerically-oriented, 27-exon-containing transcription unit that spans antisensestrand nucleotides 23,107,919-23,151,682 (NCBI36/hg18) (Figure3A) and encodes the inducible isoform of nitric oxide (NO)-synthase (NOS) 2, which produces NO, a labile uncharged lipophilic gas molecule that freely passes phospholipid bilayers.
TablesTables1 to 2are available in the Supplementary Files section