Comprehensive epigenomic profiling of human alveolar epithelial differentiation identifies key epigenetic states and transcription factor co-regulatory networks for maintenance of distal lung identity.

BACKGROUND: Disruption of alveolar epithelial cell (AEC) differentiation is implicated in distal lung diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma that impact morbidity and mortality worldwide. Elucidating underlying disease pathogenesis requires a mechanistic molecular understanding of AEC differentiation. Previous studies have focused on changes of individual transcription factors, and to date no study has comprehensively characterized the dynamic, global epigenomic alterations that facilitate this critical differentiation process in humans. RESULTS: We comprehensively profiled the epigenomic states of human AECs during type 2 to type 1-like cell differentiation, including the methylome and chromatin functional domains, and integrated this with transcriptome-wide RNA expression data. Enhancer regions were drastically altered during AEC differentiation. Transcription factor binding analysis within enhancer regions revealed diverse interactive networks with enrichment for many transcription factors, including NKX2-1 and FOXA family members, as well as transcription factors with less well characterized roles in AEC differentiation, such as members of the MEF2, TEAD, and AP1 families. Additionally, associations among transcription factors changed during differentiation, implicating a complex network of heterotrimeric complex switching in driving differentiation. Integration of AEC enhancer states with the catalog of enhancer elements in the Roadmap Epigenomics Mapping Consortium and Encyclopedia of DNA Elements (ENCODE) revealed that AECs have similar epigenomic structures to other profiled epithelial cell types, including human mammary epithelial cells (HMECs), with NKX2-1 serving as a distinguishing feature of distal lung differentiation. CONCLUSIONS: Enhancer regions are hotspots of epigenomic alteration that regulate AEC differentiation. Furthermore, the differentiation process is regulated by dynamic networks of transcription factors acting in concert, rather than individually. These findings provide a roadmap for understanding the relationship between disruption of the epigenetic state during AEC differentiation and development of lung diseases that may be therapeutically amenable.

DNA methylation profiles in diffuse large B-cell lymphoma and their relationship to gene expression status.

In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2 and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of 67 genes proximal (within 500 bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1 and FLJ21062 expression with increasing methylation suggest that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.

Migration rules: functional properties of naive and effector/memory-like regulatory T cell subsets.

Suppressor T cells were first described in the early 1970s, but since the hypothetical soluble suppressor factor could not be identified on a molecular level and since appropriate cellular markers were lacking, the suppressor T cell concept vanished for a long time. The discovery by Sakaguchi and co-workers, that the adoptive transfer of CD25+CD4+ -depleted T cells induced several organ-specific autoimmune diseases in immunodeficient recipients, put the suppressor T cell model back into the focus of many immunologists. CD25+CD4+ T cells were named regulatory T cells (Treg) and since then have been intensively characterized by many groups. It has now been well documented in a variety of models that CD25+CD4+ Tregs, in addition to cell-intrinsic peripheral tolerance mechanisms such as anergy induction and peripheral deletion, play indispensable roles in the maintenance of natural self-tolerance, in averting autoimmune responses as well as in controlling inflammatory reactions. However, a number of fundamental questions concerning their origin, mechanism of action, and the sites of suppression remain elusive and are currently a matter of debate. Notably, the potential heterogeneity of Tregs with respect to phenotype and function deserves attention and is a major issue discussed in this review.

Association tests in nuclear families.

We present a conditional likelihood approach for testing linkage disequilibrium in nuclear families having multiple affected offspring. The likelihood, conditioned on the identity-by-descent (IBD) structure of the sibling genotypes, is unaffected by familial correlation in disease status that arises from linkage between a marker locus and the unobserved trait locus. Two such conditional likelihoods are compared: one that conditions on IBD and phase of the transmitted alleles and a second which conditions only on IBD of the transmitted alleles. Under the log-additive model, the first likelihood is equivalent to the allele-counting methods proposed in the literature. The second likelihood is valid under the added assumption of equal male and female recombination fractions. In a simulation study, we demonstrated that in sibships having two or three affected siblings the score test from each likelihood had the correct test size for testing disequilibrium. They also led to equivalent power to detect linkage disequilibrium at the 5% significance level.

Clinical factors associated with progression of glaucomatous optic disc damage in treated patients.

BACKGROUND: Reducing intraocular pressure (IOP) in glaucomatous eyes does not always prevent disease progression. OBJECTIVE: To determine the clinical factors associated with progressive optic disc damage in glaucomatous eyes receiving treatment to reduce IOP. METHODS: Baseline and follow-up optic disc photographs as well as demographic and clinical data were retrospectively studied in 186 eyes of 93 patients with primary open-angle glaucoma, and in 138 eyes of 69 patients with normal-pressure glaucoma. The patients with primary open-angle glaucoma were included in the study only if their treated IOPs during a follow-up period of 5 years were less than 21 mm Hg. The patients with normal-pressure glaucoma were included only if their IOPs were reduced by at least 20% during the follow-up period. The association of progressive optic disc damage with patient- and eye-specific characteristics was examined using multivariate analysis. RESULTS: During the 5-year study period, 141 (43.5%) of the 324 eyes exhibited progressive optic disc damage defined by at least a 5% decrease in the neural rim area-to-disc area ratio. Using multivariate analysis, the following were found to be strongly associated with progressive neural rim damage: a baseline smaller neural rim area-disc area ratio (P<.001); a baseline larger zone beta area-disc area ratio (P =.04); a baseline larger parapapillary atrophy length-disc circumference ratio (P =.05); a diagnosis of normal-pressure glaucoma (P =.01); and combined medical and surgical treatment prior to the study period (P =.01). CONCLUSIONS: Clinical factors other than IOP may be important indicators of subsequent progression of glaucomatous optic disc damage. Our findings suggest that eyes with advanced glaucomatous optic disc damage and normal-pressure glaucoma are more likely to progress despite receiving treatment to reduce IOP.

Gene-environment interaction and affected sib pair linkage analysis.

OBJECTIVES: Gene-environment (GxE) interaction influences risk for many complex disease traits. However, genome screens using affected sib pair linkage techniques are typically conducted without regard for GxE interaction. We propose a simple extension of the commonly used mean test and evaluate its power for several forms of GxE interaction. METHODS: We compute expected IBD sharing by sibling exposure profile, that is by whether two sibs are exposed (EE), unexposed (UU), or are discordant for exposure (EU). We describe a simple extension of the mean test, the "mean-interaction" test that utilizes heterogeneity in IBD sharing across EE, EU, and UU sib pairs in a test for linkage. RESULTS: The mean-interaction test provides greater power than the mean test for detecting linkage in the presence of moderate or strong GxE interaction, typically when the interaction relative risk (R(ge)) exceeds 3 or is less than 1/3. In the presence of strong interaction (R(ge) = 10), the required number of affected sib pairs to achieve 80% power for detecting linkage is approximately 30% higher when the environmental factor is ignored in the mean test, than when it is utilized in the mean-interaction test. CONCLUSION: Linkage methods that incorporate environmental data and allow for interaction can lead to increased power for localizing a disease gene involved in a GxE interaction.

A case-control study of microsomal epoxide hydrolase, smoking, meat consumption, glutathione S-transferase M3, and risk of colorectal adenomas.

We estimated associations between polymorphisms in the gene encoding microsomal epoxide hydrolase (mEH) among 464 cases diagnosed with first occurrence of colorectal adenoma and 510 matched controls. In an analysis controlling only for the matching variables, we found little or no association between adenoma and mEH genotypes defined by polymorphisms at either codon 113 and 139 or mEH activity predicted by both polymorphisms. However, in subsequent analyses, high predicted mEH activity was significantly associated with adenoma among certain subgroups defined by smoking history [odds ratio (OR), 4.27; 95% confidence interval (CI), 1.68-10.81 among current smokers; interaction, P = 0.11], meat consumption (OR, 2.47; CI, 0.99-6.19 among individuals who regularly eat well-done meat; interaction, P = 0.03), and genotypes for the *A/*B polymorphism in the gene encoding glutatione S-transferase M3 (OR, 2.60; CI, 1.28-5.28 among individuals with *A*A genotype; interaction, P = 0.03). These findings are consistent with causal roles for environmental polycyclic aromatic hydrocarbons and genetically encoded variants in enzymes whose actions lead to the production of activated polycyclic aromatic hydrocarbon metabolites.

Stratified case sampling and the use of family controls.

We compare the asymptotic relative efficiency (ARE) of different study designs for estimating gene and gene-environment interaction effects using matched case-control data. In the sampling schemes considered, cases are selected differentially based on their family history of disease. Controls are selected either from unrelated subjects or from among the case's unaffected siblings and cousins. Parameters are estimated using weighted conditional logistic regression, where the likelihood contributions for each subject are weighted by the fraction of cases sampled sharing the same family history. Results showed that compared to random sampling, over-sampling cases with a positive family history increased the efficiency for estimating the main effect of a gene for sib-control designs (103-254% ARE) and decreased efficiency for cousin-control and population-control designs (68-94% ARE and 67-84% ARE, respectively). Population controls and random sampling of cases were most efficient for a recessive gene or a dominant gene with an relative risk less than 9. For estimating gene-environment interactions, over-sampling positive-family-history cases again led to increased efficiency using sib controls (111-180% ARE) and decreased efficiency using population controls (68-87% ARE). Using case-cousin pairs, the results differed based on the genetic model and the size of the interaction effect; biased sampling was only slightly more efficient than random sampling for large interaction effects under a dominant gene model (relative risk ratio = 8, 106% ARE). Overall, the most efficient study design for studying gene-environment interaction was the case-sib-control design with over-sampling of positive-family-history-cases.

Combined linkage and association analysis in pedigrees.

We applied a combined linkage and association model for quantitative traits in pedigrees to identify possible functional polymorphisms and to test for association resulting from population stratification and admixture. Functional polymorphisms are identified as variants that are significantly associated with a trait (high chi 2 value) and showing no residual evidence of linkage (low lod score). Applying our model to the simulated data in the population isolate (replicate 1) we correctly identified the polymorphism in gene 6 (MG1) that affects Q1. Without modeling association the lod score for Q1 was 5.4. At the site of the functional variant (5782 bp) the association chi 2 was 88.1 on 1 df (p < 0.001) and the lod score was 0.003. We estimated a 3.7-unit increase in the average Q1 for each extra copy of the polymorphism (95% CI = 2.95-4.41) and there was no evidence of population stratification or admixture (chi 2 = 0.08 on 2 df). For Q5 and gene 2, modeling the sequence variants at 11 loci simultaneously identified multiple functional variants. Including the main effect of 11 marker genotypes reduced the lod score at gene 2 from 8.7 to 0.9. Again, no evidence of population stratification or admixture was found (all chi 2 < 4.9 on 2 df; p > 0.05).

Should we consider gene x environment interaction in the hunt for quantitative trait loci?

We address the question of whether one can obtain increased power for finding a quantitative trait locus (QTL) if a gene x environment (G x E) interaction is incorporated directly into the linkage analysis. We consider both parametric and nonparametric analysis approaches to including G x E interaction. For the former, we utilize joint segregation and linkage analysis to estimate simultaneously the recombination fraction and a G x E interaction effect, as well as the remaining model parameters. The nonparametric approach is based on an extension of the Haseman-Elston method applied to sib pairs to include a regression of the squared trait difference on marker-identity-by-descent (IBD) probability (pi), the sibling covariate sum (z), and pi x z. We utilize 50 replicates of the simulated data and compare empirical power of the various approaches to detect MG4, a locus that is involved in a strong interaction with age for Q4 and in a weaker interaction with environmental factor E2 for Q3. Using the parametric approach, including a G x age effect does increase power for detecting linkage between MG4 and Q4 compared with ignoring the interaction (powers 58% and 38%, respectively, to exceed a lod score of 3.0). On the other hand, including a G x E2 interaction has little effect on the power to detect linkage between MG4 and Q3. The nonparametric approach leads to qualitatively similar findings. We conclude that it is beneficial to incorporate G x E interaction into a linkage analysis, provided the interaction effect is of sufficiently strong magnitude.

Hostility, social support, and carotid artery atherosclerosis in the National Heart, Lung, and Blood Institute Family Heart Study.

This cross-sectional study investigates the association of hostility and social support (measured by standardized instruments) to carotid artery atherosclerosis in men and women with a high familial risk for coronary heart disease (CHD) and those with low to medium risk. The hypothesis was that high hostility and low social support would have a stronger association in subjects with a familial predisposition to CHD. There were 535 low- to medium-risk women, 491 low- to medium-risk men, 1,950 high-risk women, and 1,667 high-risk men in the study. The extent of carotid artery atherosclerosis was assessed by B-mode ultrasound imaging. A lesion was defined as an intimal-medial far wall thickness of 1 mm in the common, internal, or carotid bifurcation, or identification of plaque at any site. Odds ratios and their 95% confidence intervals were calculated using generalized estimating equations (GEE) for logistic regression. Family was specified as the clustering variable, and robust SEEs were obtained that account for dependence of the data within families. After controlling for age, education, body mass index, ever having smoked, ever drinking > 5 drinks a day, and metabolic index, hostility was significantly associated with increased odds of carotid lesions in only high-risk women. High-risk women showed a significantly reduced odds of carotid lesions with high social support, but the extent of this protection was reduced when age and education were included in the equation. A combination of high hostility and low social support was associated with higher odds than hostility alone in both high-risk men and women. These results suggest that women with a high familial predisposition for CHD may be more vulnerable to cardiovascular influences from hostility and social support than high-risk men or men and women with low to medium risk.

Corneal scarring and vision in keratoconus: a baseline report from the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study.

PURPOSE: The multicenter Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study is a prospective, observational study of 1,209 keratoconus patients. We report on the correlation of corneal scarring with clinical and patient-reported variables at the baseline visit. METHODS: Patients completed a questionnaire on their vision, effect of glare, contact lens wear, and work-related issues. Clinical examination included high- and low-contrast visual acuity, refraction, assessment of corneal scarring by the clinician and by photography, and measurement of corneal curvature. The correlation of central corneal scarring with visual acuity and patient-reported variables was analyzed using multiple regression analysis and generalized estimating equations. RESULTS: High- and low-contrast visual acuity with habitual and optimal correction is reduced in scarred eyes. Multiple regression analyses controlling for age, contact lens wear, and disease severity show that central scarring is associated with poorer visual acuity and increased patient-reported symptoms of glare. Restrictions on day-to-day activities do not appear to be associated with corneal scarring above and beyond the effects of keratoconus alone. CONCLUSIONS: Corneal scarring in keratoconus is significantly associated with decreased high- and low-contrast visual acuity.

Factors associated with corneal scarring in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study.

PURPOSE: The multicenter Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study is a prospective, observational study of 1,209 keratoconus patients. We report on the factors associated with corneal scarring at baseline. METHODS: We defined corneal scarring as scars that had been detected both by the clinician examining the patient with the slit-lamp biomicroscope and by masked readers of corneal photographs at the CLEK Photography Reading Center. We investigated associations between corneal scarring and patient variables including gender, ethnicity, a family history of keratoconus, a history of ocular trauma, eye rubbing, contact lens wear, rigid contact lens fitting relationships, and corneal findings (such as curvature, Vogt's striae, Fleischer's ring, and central/apical staining). Multiple logistic regression analysis using generalized estimating equations to adjust for the correlation between eyes was used for analysis. RESULTS: The following factors were found to increase the odds of corneal scarring at baseline in the CLEK Study: corneal staining (odds ratios (OR) = 3.40, 95% confidence interval 2.53-4.59), contact lens wear (OR = 3.51, 95% confidence interval 2.27-5.45), Fleischer's ring (OR = 1.63, 95% confidence interval 1.11-2.40), steeper first definite apical clearance lens base curve radius (per diopter, OR = 1.29, 95% confidence interval 1.25-1.33), and age (per decade, OR = 1.54, 95% confidence interval 1.35-1.75). CONCLUSIONS: These baseline data suggest that corneal scarring in keratoconus is associated with corneal staining, contact lens wear, Fleischer's ring, a steeper cornea, and increasing age. The factors that imply added risk for corneal scarring that may be affected by practitioner intervention are staining of the cornea, contact lens wear, and the contact lens fitting relationship.

The methylenetetrahydrofolate reductase 677C-->T polymorphism and distal colorectal adenoma risk.

A common polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene, where a cytosine at nucleotide 677 is replaced by a thymine (677C-->T), is associated with enzyme thermolability and a reduction in the conversion of 5,10-methyltetrahydrofolate (5,10-MTHF) into 5-methyltetrahydrofolate. We assessed the association between homozygosity for the MTHFR 677CT genotype (TT) and colorectal adenoma risk in a large sigmoidoscopy-based case-control study of members of a prepaid health plan in Los Angeles. MTHFR genotype was determined for 471 cases and 510 age-, sex-, clinic-, and sigmoidoscopy-date-matched controls. Information on RBC and plasma folate levels were analyzed for 331 cases and 350 controls. When compared with the presence of at least one wild-type allele (CT/CC), the odds ratio (OR) for the TT genotype was 1.19 [95% confidence interval (CI), 0.77-1.76] after adjusting for race and the matching factors. Compared with those in the lowest quartiles of RBC and plasma folate and a wild-type allele, adenoma risk was increased for TT homozygotes in the lowest folate quartiles (genotype: OR, 2.04 and 95% CI, 0.6-7.0; OR, 1.84 and 95% CI, 0.6-7.0 for RBCs and plasma folate, respectively) and decreased in TT homozygotes in the highest quartiles (genotype: OR, 0.82 and 95% CI, 0.32-2.10; OR, 0.65 and 95% CI, 0.22-1.95, respectively). There was also a significant interaction between TT genotype and the increased adenoma risk associated with alcohol. These data are consistent with an interaction between MTHFR genotype and folate availability.

Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron-sulfur proteins.

The assembly of iron-sulfur (Fe/S) clusters in a living cell is mediated by a complex machinery which, in eukaryotes, is localised within mitochondria. Here, we report on a new component of this machinery, the protein Isa2p of the yeast Saccharomyces cerevisiae. The protein shares sequence similarity with yeast Isa1p and the bacterial IscA proteins which recently have been shown to perform a function in Fe/S cluster biosynthesis. Like the Isa1p homologue, Isa2p is localised in the mitochondrial matrix as a soluble protein. Deletion of the ISA2 gene results in the loss of mitochondrial DNA and a strong growth defect. Simultaneous deletion of the ISA1 gene does not further exacerbate this growth phenotype suggesting that the Isa proteins perform a non-essential function. When Isa2p was depleted by regulated gene expression, mtDNA was maintained, but cells grew slowly on non-fermentable carbon sources. The maturation of both mitochondrial and cytosolic Fe/S proteins was strongly impaired in the absence of Isa2p. Thus, Isa2p is a new member of the Fe/S cluster biosynthesis machinery of the mitochondrial matrix and may be involved in the binding of an intermediate of Fe/S cluster assembly.

Testing linkage disequilibrium in sibships.

We describe the use of multivariate regression for testing allelic association in the presence of linkage, using marker genotype data from sibships. The test is valid, provided that the correct mean structure is modeled but does not require the correlation structure within families to be specified. The test can be implemented using standard statistical software such as the SAS programming language. In a simulation study, we evaluated this new test in comparison with one from a standard, matched-case-control analysis. First, we noted that the genetic effect needed to be quite extreme before residual familial correlation due to linkage led to false inference using the standard, matched-pair analysis. Second, we showed that under examples of extreme residual familial correlation, the new test had the correct test size. Third, we found that the test was more powerful than the sibship disequilibrium test of Horvath and Laird. Finally, we concluded that although the standard analysis may lead to correct inference for practical purposes, the new test is valid, even under extreme residual familial correlation and with no cost in power at the causal locus.

Linkage analysis for diseases with variable age of onset.

We present a method for the multivariate linkage analysis of the age of onset of a disease. The approach allows the incorporation of covariates for the study of gene by environment interactions. It is applicable to general pedigrees. The likelihood of the data is expressed as a function of the number of alleles identical by descent at a marker, the censored ages of onset and disease status, and environmental exposures. In a simulation study, we compare the power to detect linkage under different sampling schemes for either a dominant or recessive trait when approximately 10% of individuals are gene carriers. The majority of the linkage information from a sample of randomly selected sib pairs was retained when the analyses were limited to sibships with one sibling having early-onset disease (<59 years old). Incorporating parental phenotypes could improve the power to detect the gene. When the sample consists of affected sib pairs (ASPs) having variable age of onset, the likelihood ratio (LR) test had higher power than the means (t(2)) test for detecting a locus with a large genetic relative risk (R(g) = 20). However, the power of the two tests was similar when ASPs are selected so that the proband has an early onset of disease. Lastly, the LR test had more power than the t(2) test to detect linkage in the presence of gene by environment interactions.

A joint test of linkage and gene x environment interaction, with affected sib pairs.

In the presence of gene x environment (G x E) interaction, the expected proportion of alleles shared identical by descent at a linked marker locus by a pair of affected sibs depends on the exposure profile of the two sibs, i.e., whether both are exposed to E, only one is exposed, or neither are exposed. In this paper, we propose an extension of the commonly used mean test of linkage to test for differential identical-by-descent (IBD) sharing across sib-exposure profiles. The method can be viewed as a test for linkage in the presence of G x E interaction, or as a test for G x E interaction in the presence of linkage. Applied to the simulated GAW11 data, our method successfully localized disease locus C and its interactive relationship with environmental factor E1. At the 5% significance level, use of our method led to increased power to detect linkage (56%) to this disease locus compared to use of the standard mean test (32%); at the 0.001 significance level, the corresponding power estimates were 20% and 4%, respectively. For a gene that interacts with an environmental factor, we conclude that use of the environmental factor in linkage analysis can improve detection rates while also providing information about underlying mechanisms.

Association tests using unaffected-sibling versus pseudo-sibling controls.

We used family-matched case-control data to screen the genome for markers associated with disease in the simulated data set. Two different types of controls were considered: (1) unaffected siblings and (2) 'pseudo siblings,' a comparison sample created using the parental alleles. The scans were conducted on the first replicate of each study population. Overall, the two methods identified 14 marker loci associated with disease at the 0.001 significance level. Marker D1G24 (locus D) was the only true disease locus found by both approaches. No associations were found at any of the markers flanking the unobserved disease susceptibility loci (A, B, or C). We subsequently pooled the 25 replicates from a single population. This large sample still did not yield any associations at the flanking markers. We tested for association at locus D using a pseudo-sib approach restricted to alleles shared identical by descent between affected sib pairs. The power was 44% (11/25 replicates) at a significance level of 0.001.

A generalized estimating equations approach to linkage analysis in sibships in relation to multiple markers and exposure factors.

We describe a multiple regression approach to nonparametric linkage analysis in sibships incorporating multiple genetic loci, environmental covariates, and interactions. The covariance in trait residuals between sib pairs is treated as the dependent variable, regressed upon identical-by-descent sharing probabilities and interaction effects, using generalized estimating equations to allow for the correlations among multiple sib pairs within a sibship. Individual covariates can also be introduced in the model for the trait means. An application to the GAW11 simulated data revealed linkage with each of the four simulated loci, as well as gene x environment interactions of E1 with loci C and D and gene x gene interactions among the cluster of loci A, B, and D.