Methodological Considerations in Estimation of Phenotype Heritability Using Genome-Wide SNP Data, Illustrated by an Analysis of the Heritability of Height in a Large Sample of African Ancestry Adults

Height has an extremely polygenic pattern of inheritance. Genome-wide association studies (GWAS) have revealed hundreds of common variants that are associated with human height at genome-wide levels of significance. However, only a small fraction of phenotypic variation can be explained by the aggregate of these common variants. In a large study of African-American men and women (n = 14,419), we genotyped and analyzed 966,578 autosomal SNPs across the entire genome using a linear mixed model variance components approach implemented in the program GCTA (Yang et al Nat Genet 2010), and estimated an additive heritability of 44.7% (se: 3.7%) for this phenotype in a sample of evidently unrelated individuals. While this estimated value is similar to that given by Yang et al in their analyses, we remain concerned about two related issues: (1) whether in the complete absence of hidden relatedness, variance components methods have adequate power to estimate heritability when a very large number of SNPs are used in the analysis; and (2) whether estimation of heritability may be biased, in real studies, by low levels of residual hidden relatedness. We addressed the first question in a semi-analytic fashion by directly simulating the distribution of the score statistic for a test of zero heritability with and without low levels of relatedness. The second question was addressed by a very careful comparison of the behavior of estimated heritability for both observed (self-reported) height and simulated phenotypes compared to imputation R2 as a function of the number of SNPs used in the analysis. These simulations help to address the important question about whether today's GWAS SNPs will remain useful for imputing causal variants that are discovered using very large sample sizes in future studies of height, or whether the causal variants themselves will need to be genotyped de novo in order to build a prediction model that ultimately captures a large fraction of the variability of height, and by implication other complex phenotypes. Our overall conclusions are that when study sizes are quite large (5,000 or so) the additive heritability estimate for height is not apparently biased upwards using the linear mixed model; however there is evidence in our simulation that a very large number of causal variants (many thousands) each with very small effect on phenotypic variance will need to be discovered to fill the gap between the heritability explained by known versus unknown causal variants. We conclude that today's GWAS data will remain useful in the future for causal variant prediction, but that finding the causal variants that need to be predicted may be extremely laborious

Methodological Considerations in Estimation of Phenotype Heritability Using Genome-Wide SNP Data, Illustrated by an Analysis of the Heritability of Height in a Large Sample of African Ancestry Adults

Height has an extremely polygenic pattern of inheritance. Genome-wide association studies (GWAS) have revealed hundreds of common variants that are associated with human height at genome-wide levels of significance. However, only a small fraction of phenotypic variation can be explained by the aggregate of these common variants. In a large study of African-American men and women (n = 14,419), we genotyped and analyzed 966,578 autosomal SNPs across the entire genome using a linear mixed model variance components approach implemented in the program GCTA (Yang et al Nat Genet 2010), and estimated an additive heritability of 44.7% (se: 3.7%) for this phenotype in a sample of evidently unrelated individuals. While this estimated value is similar to that given by Yang et al in their analyses, we remain concerned about two related issues: (1) whether in the complete absence of hidden relatedness, variance components methods have adequate power to estimate heritability when a very large number of SNPs are used in the analysis; and (2) whether estimation of heritability may be biased, in real studies, by low levels of residual hidden relatedness. We addressed the first question in a semi-analytic fashion by directly simulating the distribution of the score statistic for a test of zero heritability with and without low levels of relatedness. The second question was addressed by a very careful comparison of the behavior of estimated heritability for both observed (self-reported) height and simulated phenotypes compared to imputation R2 as a function of the number of SNPs used in the analysis. These simulations help to address the important question about whether today's GWAS SNPs will remain useful for imputing causal variants that are discovered using very large sample sizes in future studies of height, or whether the causal variants themselves will need to be genotyped de novo in order to build a prediction model that ultimately captures a large fraction of the variability of height, and by implication other complex phenotypes. Our overall conclusions are that when study sizes are quite large (5,000 or so) the additive heritability estimate for height is not apparently biased upwards using the linear mixed model; however there is evidence in our simulation that a very large number of causal variants (many thousands) each with very small effect on phenotypic variance will need to be discovered to fill the gap between the heritability explained by known versus unknown causal variants. We conclude that today's GWAS data will remain useful in the future for causal variant prediction, but that finding the causal variants that need to be predicted may be extremely laborious

Characterization of UreR interaction with the urea- and UreR-dependent promoters of the urease gene clusters

Urease, is a primary virulence factor of organisms infecting the urinary tract and gastroduodenal region. Urease catalyzes the hydrolysis of urea to release carbon dioxide and two molecules of ammonia. Within Enterobacteriaceae , genes encoding for urease can be found either on the chromosome or on large plasmids. Urease can be either expressed constitutively, induced under nitrogen starvation, or induced by urea. The plasmid-encoded gene cluster found in organisms such as Providencia stuartii and the chromosomally-encoded gene cluster of Proteus mirabilis are regulated by urea. These loci share similar genetic organization with the genes ureDABCEFG encoding for the structural subunits and accessory proteins of the enzyme. The divergently transcribed ureR encodes for a regulatory protein. Three promoters (ureRp, Dp, and Gp) are active in the presence of urea and UreR. The divergently transcribed ureRp and ureDp are situated within a 414 bp and 494 bp ureRp-Dp intergenic region of the plasmid-encoded and P. mirabilis gene clusters respectively. The minimal promoter sequence requirement for full UreR- and urea-dependent activation of ureRp and ureDp was localized to the 147 bp separating the ureR and ureD transcription start sites in the plasmid-encoded gene cluster. P. mirabilis contains a similar region of 145 bps. -10 and -35 consensus RNA polymerase binding hexamers relative to the start of ureR transcription and a -10 consensus hexamer relative to the start of ureD transcription were identified.UreR is an AraC-like transcriptional activator essential for urea-induced activation of the urease gene cluster. UreR from the plasmid-encoded and the P. mirabilis urease gene clusters share 70% amino acid identity and are functionally interchangeable. To elucidate the role of UreR in the urea-induced activation of the urease gene cluster, a purified recombinant (rUreR) protein from the plasmid-encoded gene cluster was isolated and assessed for DNA binding capability in the presence and absence of urea. Electrophoretic mobility shift assays and DNase I footprint analysis reveal that rUreR can bind specifically to the ureRp and ureDp from the plasmid-encoded and P. mirabilis urease gene clusters. In contrast, ureGp was not bound by rUreR under similar reaction conditions. Promoter interaction of rUreR is independent of urea. However, addition of urea increased the DNA interaction by 2-fold. Comparison of the DNA binding sites did not reveal a clear consensus sequence. A 13 bp sequence of TPuPyNNPyTNNATTG was identified -46 to -34 relative to the start of ureR transcription and -49 to -37 relative to the start of ureD transcription. Dissociation rate constants, which could only be determined in the presence of urea, revealed that the ureDp site has a higher affinity for rUreR. ureRp demonstrated preferential binding to RNA polymerase in vitro. In addition, specific transcription from the ureD promoter was observed only in the presence of rUreR and urea. These data demonstrate that UreR mediates transcription by direct interaction with the ureD and ureR promoters. Urea might enhance the UreR-dependent regulation of the urease gene cluster by stabilizing the UreR-DNA interaction

Anaplasma phagocytophilum Modulates gp91(phox) Gene Expression through Altered Interferon Regulatory Factor 1 and PU.1 Levels and Binding of CCAAT Displacement Protein

Infection of neutrophil precursors with Anaplasma phagocytophilum, the causative agent of human granulocytic ehrlichiosis, results in downregulation of the gp91(phox) gene, a key component of NADPH oxidase. We now show that repression of gp91(phox) gene transcription is associated with reduced expression of interferon regulatory factor 1 (IRF-1) and PU.1 in nuclear extracts of A. phagocytophilum-infected cells. Loss of PU.1 and IRF-1 correlated with increased binding of the repressor, CCAAT displacement protein (CDP), to the promoter of the gp91(phox) gene. Reduced protein expression of IRF-1 was observed with or without gamma interferon (IFN-γ) stimulation, and the defect in IFN-γ signaling was associated with diminished binding of phosphorylated Stat1 to the Stat1 binding element of the IRF-1 promoter. The diminished levels of activator proteins and enhanced binding of CDP account for the transcriptional inhibition of the gp91(phox) gene during A. phagocytophilum infection, providing evidence of the first molecular mechanism that a pathogen uses to alter the regulation of genes that contribute to an effective respiratory burst

Abstract 3204: Association between oxidative damage and early adverse skin reactions following postoperative adjuvant radiotherapy in breast cancer patients

Abstract Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer death in American women. Lumpectomy followed by radiotherapy (RT) has significantly improved survival. However, about 30% of patients develop a grade 2 or worse early or late skin reaction, pain, breast edema and poor cosmetic results that impact quality of life. Inter-individual variability in the development of RT-induced adverse reactions in normal tissue is well-documented for both acute and late effects. Therefore, identifying individuals which may have severe reactions could assist the radiation oncologist in tailoring a treatment regimen which provides adequate care with minimal undesirable effects. One of the hallmarks of radiation induced DNA damage is the oxidation of the nucleotide guanine. Repair and excretion of this modified base known as 8-hydroxydeoxyguanosine (8-OHdG) serves as a biomarker of oxidative stress and DNA damage. In an ongoing study, we investigated the urinary level of 8-OHdG (presented as ng/mg creatinine) in urine of the first 103 breast cancer patients undergoing adjuvant RT following breast conservation surgery. The normal skin toxicity at 3 weeks and at the end of treatment was assessed and associated with 8-OHdG levels. There was a significant correlation between pre- (55.8+35.2) and post-RT (55.5+37.4) urinary 8-OHdG levels (Pearson correlation coefficient=0.44; p&amp;lt;0.001). Within our patient population, Non-Hispanic Whites had a significantly higher 8-OHdG level (80.9+52.6) compared to Hispanic Whites (48.2+25.2, p=0.001) and Black or African Americans (43.7+20.9, p=0.008). With limited sample size, at 3 weeks of post-RT, patients with no apparent skin reaction showed a non-significantly lower pre-RT 8-OHdG (46.4+23.5) compared to patients with a grade 1 or 2 skin toxicity (57.8+37.5, p=0.18). Similar trend was also observed for post-RT 8-OHdG (45.6+20.7 vs. 57.7+40.3; p=.20). At the end of RT, patients with detectable skin toxicity had a baseline 8-OHdG level of 56.0+35.6 while patients without a skin reaction had a baseline level of 39.6+17.6. In summary, our preliminary data with limited sample size showed significant racial/ethnic differences in urinary 8-OHdG levels and promising but not significant association between 8-OHdG levels and skin toxicity grade. Larger studies are warranted to validate these interesting findings. The outcome of the ongoing study (n=1000) will target effective intervention and treatment strategies, and ultimately improve quality of life and progression-free survival in high-risk breast cancer patient populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2011-3204</jats:p

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice

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