A clustering approach to improve our understanding of the genetic and phenotypic complexity of chronic kidney disease.

Chronic kidney disease (CKD) is a complex disorder that causes a gradual loss of kidney function, affecting approximately 9.1% of the world's population. Here, we use a soft-clustering algorithm to deconstruct its genetic heterogeneity. First, we selected 322 CKD-associated independent genetic variants from published genome-wide association studies (GWAS) and added association results for 229 traits from the GWAS catalog. We then applied nonnegative matrix factorization (NMF) to discover overlapping clusters of related traits and variants. We computed cluster-specific polygenic scores and validated each cluster with a phenome-wide association study (PheWAS) on the BioMe biobank (n=31,701). NMF identified nine clusters that reflect different aspects of CKD, with the top-weighted traits signifying areas such as kidney function, type 2 diabetes (T2D), and body weight. For most clusters, the top-weighted traits were confirmed in the PheWAS analysis. Results were found to be more significant in the cross-ancestry analysis, although significant ancestry-specific associations were also identified. While all alleles were associated with a decreased kidney function, associations with CKD-related diseases (e.g., T2D) were found only for a smaller subset of variants and differed across genetic ancestry groups. Our findings leverage genetics to gain insights into the underlying biology of CKD and investigate population-specific associations.

Trans-ethnic analysis of metabochip data identifies two new loci associated with BMI.

OBJECTIVE: Body mass index (BMI) is commonly used to assess obesity, which is associated with numerous diseases and negative health outcomes. BMI has been shown to be a heritable, polygenic trait, with close to 100 loci previously identified and replicated in multiple populations. We aim to replicate known BMI loci and identify novel associations in a trans-ethnic study population. SUBJECTS: Using eligible participants from the Population Architecture using Genomics and Epidemiology consortium, we conducted a trans-ethnic meta-analysis of 102 514 African Americans, Hispanics, Asian/Native Hawaiian, Native Americans and European Americans. Participants were genotyped on over 200 000 SNPs on the Illumina Metabochip custom array, or imputed into the 1000 Genomes Project (Phase I). Linear regression of the natural log of BMI, adjusting for age, sex, study site (if applicable), and ancestry principal components, was conducted for each race/ethnicity within each study cohort. Race/ethnicity-specific, and combined meta-analyses used fixed-effects models. RESULTS: We replicated 15 of 21 BMI loci included on the Metabochip, and identified two novel BMI loci at 1q41 (rs2820436) and 2q31.1 (rs10930502) at the Metabochip-wide significance threshold (P<2.5 × 10-7). Bioinformatic functional investigation of SNPs at these loci suggests a possible impact on pathways that regulate metabolism and adipose tissue. CONCLUSION: Conducting studies in genetically diverse populations continues to be a valuable strategy for replicating known loci and uncovering novel BMI associations.

Implementing Algorithm-Guided Warfarin Dosing in an Ethnically Diverse Patient Population Using Electronic Health Records and Preemptive CYP2C9 and VKORC1 Genetic Testing.

Implementation of pharmacogenetic-guided warfarin dosing has been hindered by inconsistent results from reported clinical trials and a lack of available algorithms that include alleles prevalent in non-white populations. However, current evidence indicates that algorithm-guided dosing is more accurate than empirical dosing. To facilitate multiethnic algorithm-guided warfarin dosing using preemptive genetic testing, we developed a strategy that accounts for the complexity of race and leverages electronic health records for algorithm variables and deploying point-of-care dose recommendations.

Genetic variation among 82 pharmacogenes: The PGRNseq data from the eMERGE network.

Genetic variation can affect drug response in multiple ways, although it remains unclear how rare genetic variants affect drug response. The electronic Medical Records and Genomics (eMERGE) Network, collaborating with the Pharmacogenomics Research Network, began eMERGE-PGx, a targeted sequencing study to assess genetic variation in 82 pharmacogenes critical for implementation of "precision medicine." The February 2015 eMERGE-PGx data release includes sequence-derived data from ∼5,000 clinical subjects. We present the variant frequency spectrum categorized by variant type, ancestry, and predicted function. We found 95.12% of genes have variants with a scaled Combined Annotation-Dependent Depletion score above 20, and 96.19% of all samples had one or more Clinical Pharmacogenetics Implementation Consortium Level A actionable variants. These data highlight the distribution and scope of genetic variation in relevant pharmacogenes, identifying challenges associated with implementing clinical sequencing for drug treatment at a broader level, underscoring the importance for multifaceted research in the execution of precision medicine.

Determining the effects and challenges of incorporating genetic testing into primary care management of hypertensive patients with African ancestry.

People of African ancestry (Blacks) have increased risk of kidney failure due to numerous socioeconomic, environmental, and clinical factors. Two variants in the APOL1 gene are now thought to account for much of the racial disparity associated with hypertensive kidney failure in Blacks. However, this knowledge has not been translated into clinical care to help improve patient outcomes and address disparities. GUARDD is a randomized trial to evaluate the effects and challenges of incorporating genetic risk information into primary care. Hypertensive, non-diabetic, adults with self-reported African ancestry, without kidney dysfunction, are recruited from diverse clinical settings and randomized to undergo APOL1 genetic testing at baseline (intervention) or at one year (waitlist control). Providers are educated about genomics and APOL1. Guided by a genetic counselor, trained staff return APOL1 results to patients and provide low-literacy educational materials. Real-time clinical decision support tools alert clinicians of their patients' APOL1 results and associated risk status at the point of care. Our academic-community-clinical partnership designed a study to generate information about the impact of genetic risk information on patient care (blood pressure and renal surveillance) and on patient and provider knowledge, attitudes, beliefs, and behaviors. GUARDD will help establish the effective implementation of APOL1 risk-informed management of hypertensive patients at high risk of CKD, and will provide a robust framework for future endeavors to implement genomic medicine in diverse clinical practices. It will also add to the important dialog about factors that contribute to and may help eliminate racial disparities in kidney disease.

A genome-wide association study identifies variants in KCNIP4 associated with ACE inhibitor-induced cough.

The most common side effect of angiotensin-converting enzyme inhibitor (ACEi) drugs is cough. We conducted a genome-wide association study (GWAS) of ACEi-induced cough among 7080 subjects of diverse ancestries in the Electronic Medical Records and Genomics (eMERGE) network. Cases were subjects diagnosed with ACEi-induced cough. Controls were subjects with at least 6 months of ACEi use and no cough. A GWAS (1595 cases and 5485 controls) identified associations on chromosome 4 in an intron of KCNIP4. The strongest association was at rs145489027 (minor allele frequency=0.33, odds ratio (OR)=1.3 (95% confidence interval (CI): 1.2-1.4), P=1.0 × 10(-8)). Replication for six single-nucleotide polymorphisms (SNPs) in KCNIP4 was tested in a second eMERGE population (n=926) and in the Genetics of Diabetes Audit and Research in Tayside, Scotland (GoDARTS) cohort (n=4309). Replication was observed at rs7675300 (OR=1.32 (1.01-1.70), P=0.04) in eMERGE and at rs16870989 and rs1495509 (OR=1.15 (1.01-1.30), P=0.03 for both) in GoDARTS. The combined association at rs1495509 was significant (OR=1.23 (1.15-1.32), P=1.9 × 10(-9)). These results indicate that SNPs in KCNIP4 may modulate ACEi-induced cough risk.

Genetic variation in the HLA region is associated with susceptibility to herpes zoster.

Herpes zoster, commonly referred to as shingles, is caused by the varicella zoster virus (VZV). VZV initially manifests as chicken pox, most commonly in childhood, can remain asymptomatically latent in nerve tissues for many years and often re-emerges as shingles. Although reactivation may be related to immune suppression, aging and female sex, most inter-individual variability in re-emergence risk has not been explained to date. We performed a genome-wide association analyses in 22,981 participants (2280 shingles cases) from the electronic Medical Records and Genomics Network. Using Cox survival and logistic regression, we identified a genomic region in the combined and European ancestry groups that has an age of onset effect reaching genome-wide significance (P>1.0 × 10(-8)). This region tags the non-coding gene HCP5 (HLA Complex P5) in the major histocompatibility complex. This gene is an endogenous retrovirus and likely influences viral activity through regulatory functions. Variants in this genetic region are known to be associated with delay in development of AIDS in people infected by HIV. Our study provides further suggestion that this region may have a critical role in viral suppression and could potentially harbor a clinically actionable variant for the shingles vaccine.

Design and anticipated outcomes of the eMERGE-PGx project: a multicenter pilot for preemptive pharmacogenomics in electronic health record systems.

We describe here the design and initial implementation of the eMERGE-PGx project. eMERGE-PGx, a partnership of the Electronic Medical Records and Genomics Network and the Pharmacogenomics Research Network, has three objectives: (i) to deploy PGRNseq, a next-generation sequencing platform assessing sequence variation in 84 proposed pharmacogenes, in nearly 9,000 patients likely to be prescribed drugs of interest in a 1- to 3-year time frame across several clinical sites; (ii) to integrate well-established clinically validated pharmacogenetic genotypes into the electronic health record with associated clinical decision support and to assess process and clinical outcomes of implementation; and (iii) to develop a repository of pharmacogenetic variants of unknown significance linked to a repository of electronic health record-based clinical phenotype data for ongoing pharmacogenomics discovery. We describe site-specific project implementation and anticipated products, including genetic variant and phenotype data repositories, novel variant association studies, clinical decision support modules, clinical and process outcomes, approaches to managing incidental findings, and patient and clinician education methods.

Apoptosis in podocytes induced by TGF-beta and Smad7.

Primary and secondary forms of focal segmental glomerulosclerosis (FSGS) are characterized by depletion of podocytes and constitute a central manifestation of chronic progressive glomerular diseases. Here we report that podocytes undergo apoptosis at early stages in the course of progressive glomerulosclerosis in TGF-beta1 transgenic mice. Apoptosis is associated with progressive depletion of podocytes and precedes mesangial expansion. Smad7 protein expression is strongly induced specifically in damaged podocytes of transgenic mice and in cultured murine podocytes treated with TGF-beta. TGF-beta1 and Smad7 each induce apoptosis in podocytes, and their coexpression has an additive effect. Activation of p38 MAP kinase and caspase-3 is required for TGF-beta-mediated apoptosis, but not for apoptosis induced by Smad7. Unlike TGF-beta, Smad7 inhibits nuclear translocation and transcriptional activity of the cell survival factor NF-kappaB. Our results suggest a novel functional role for Smad7 as amplifier of TGF-beta-induced apoptosis in podocytes and a new pathomechanism for podocyte depletion in progressive glomerulosclerosis.

Is Smad3 a major player in signal transduction pathways leading to fibrogenesis?

Transforming growth factor (TGF)-beta plays a central role in fibrosis, contributing both to the influx and activation of inflammatory cells, as well as to activation of fibroblasts to elaborate extracellular matrix. In the past few years, new insight has been gained into signal transduction pathways downstream of the TGF-beta receptor serine-threonine kinases with the identification of a family of evolutionarily conserved Smad proteins. Two receptor-activated Smad proteins, Smad2 and Smad3, are phosphorylated by the activated TGF-beta type I receptor kinase, after which they partner with the common mediator, Smad4, and are translocated to the nucleus to where they participate in transcriptional complexes to control expression of target genes. We have shown in wound healing studies of mice null for Smad3, that loss of this key signaling intermediate interferes with the chemotaxis of inflammatory cells to TGF-beta as well as with their ability to autoinduce TGF-beta. Moreover, studies with mouse embryo fibroblasts null for Smad3 show that TGF-beta-dependent induction of c-Jun and c-Fos, important in induction of collagen as well as in autoinduction of TGF-beta, is mediated by Smad3. Based on these observations, we hypothesize that loss of Smad3 will confer resistance to fibrosis and result in reduced inflammatory cell infiltrates, reduced autoinduction of TGF-beta, important to sustain the process, and reduced elaboration of collagen. Preliminary observations in a model of radiation-induced fibrosis confirm this hypothesis and suggest that inhibitors of Smad3 might have clinical application both to improve wound healing and to reduce fibrosis.

TGF-beta signaling in mammary gland development and tumorigenesis.

Ligands of the TGF-beta superfamily are unique in that they signal through transmembrane receptor serine-threonine kinases, rather than tyrosine kinases. The receptor complex couples to a signal transduction pathway involving a novel family of proteins, the Smads. On phosphorylation, Smads translocate to the nucleus where they modulate transcriptional responses. However, TGF-betas can also activate the mitogen-activated protein kinase (MAPK)4 pathway, and the different biological responses to TGF-beta depend to varying degrees on activation of either or both of these two pathways. The Smad pathway is a nexus for cross-talk with other signal transduction pathways and for modulation by many different interacting proteins. Despite compelling evidence that TGF-beta has tumor suppressor activity in the mammary gland, neither TGF-beta receptors nor Smads are genetically inactivated in human breast cancer, though receptor expression is reduced. Possible reasons are discussed in relation to the dual role of TGF-beta as tumor suppressor and oncogene.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-beta.

Epithelial-mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-beta (TGF-beta) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-beta-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-beta within 4 hours after treatment. TGF-beta-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell-matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-beta. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.

A prominent pathway of transforming growth factor (TGF)-beta signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-beta1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-beta1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-beta1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [(3)H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-beta1. Lack of Smad2 or Smad3 expression did not affect TGF-beta1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-beta1. Moreover, TGF-beta1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-beta1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-beta-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)(4)-Lux reporter by TGF-beta1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-beta1 signaling.

Caveolin-1 regulates transforming growth factor (TGF)-beta/SMAD signaling through an interaction with the TGF-beta type I receptor.

Transforming growth factor-beta (TGF-beta) signaling proceeds from the cell membrane to the nucleus through the cooperation of the type I and II serine/threonine kinase receptors and their downstream SMAD effectors. Although various regulatory proteins affecting TGF-beta-mediated events have been described, relatively little is known about receptor interactions at the level of the plasma membrane. Caveolae are cholesterol-rich membrane microdomains that, along with their marker protein caveolin-1 (Cav-1), have been implicated in the compartmentalization and regulation of certain signaling events. Here, we demonstrate that specific components of the TGF-beta cascade are associated with caveolin-1 in caveolae and that Cav-1 interacts with the Type I TGF-beta receptor. Additionally, Cav-1 is able to suppress TGF-beta-mediated phosphorylation of Smad-2 and subsequent downstream events. We localize the Type I TGF-beta receptor interaction to the scaffolding domain of Cav-1 and show that it occurs in a physiologically relevant time frame, acting to rapidly dampen signaling initiated by the TGF-beta receptor complex.

Smad proteins and transforming growth factor-beta signaling.

It is now generally accepted that transforming growth factor-beta (TGF-beta) has an important role in the pathogenesis of both acute and chronic forms of renal disease. Although TGF-beta's potent fibrogenic activity is considered a major factor in chronic progression of renal disease, this cytokine participates in the control of several fundamental cellular responses in the kidney including inflammation, programmed cell death, cell growth, cell differentiation, and cellular hypertrophy. Recent identification of Smad proteins as intracellular mediators of TGF-beta signaling has provided important insights into mechanisms that may determine the specificity of TGF-beta action in different renal and inflammatory cells. Thus, Smads are characterized by an astonishingly complex array of molecular and functional interactions with other signaling pathways. These emerging patterns of signaling cross talk involving Smad proteins suggest a dynamic profile of positive or negative transmodulation of TGF-beta signaling, depending on the cellular context. Understanding the interplay between these signaling cascades is an important field of investigation that will ultimately reveal new targets for precise and selective modulation of TGF-beta's diverse actions in renal diseases.

Dietary fat and reduced levels of TGFbeta1 act synergistically to promote activation of the vascular endothelium and formation of lipid lesions.

Transforming growth factor-(beta) (TGF(beta)) has a wide range of activities on vascular cells and inflammatory cells, suggesting it may have different functions during various stages of atherogenesis. We report that mice heterozygous for the deletion of the tgfb1 gene (tgfb1(+/-) mice) have reduced levels of TGF(beta)1 in the artery wall until at least 8 weeks of age. On a normal mouse chow diet, the vascular endothelium of tgfb1(+/-) mice is indistinguishable from wild-type littermates, assessed by morphology and intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. In contrast, levels of the smooth muscle isoforms of actin and myosin in medial smooth muscle cells of tgfb1(+/-) mice are significantly reduced. Following feeding a cholesterol-enriched diet for 12 weeks, high levels of ICAM-1 and VCAM-1 were detected in the vascular endothelial cells of tgfb1(+/-) mice, but not wild-type mice. Furthermore, marked deposition of lipid into the artery wall was only observed in the tgfb1(+/-) mice on the cholesterol-enriched diet. These vascular lipid lesions were accompanied by local invasion of macrophages. We conclude that deletion of a single allele of the tgfb1 gene results in a reduced level of TGFbeta1 antigen in the aorta together with reduced smooth muscle cell differentiation, whereas the addition of a high fat dietary challenge is required to activate the vascular endothelium and to promote the formation of fatty streaks resembling early atherosclerosis in humans.

Defects in TGF-beta signaling overcome senescence of mouse keratinocytes expressing v-Ha-ras.

Previous studies have shown that TGFbeta1 expression is upregulated in mouse keratinocytes infected with a v-rasHa retrovirus, although the functional significance of this has not been clear. Here we show that v-rasHa retrovirus transduced primary mouse keratinocytes undergo hyperproliferation followed by a TGFbeta1 dependent G1 growth arrest and senescence. The growth arrest is accompanied by a 15-fold increase in total TGFbeta1 secreted and a fourfold increase in secreted active TGFbeta1. When cultured in the presence of a neutralizing antibody to TGFbeta1, the senescence response is suppressed. Levels of the TGFbeta1 target p15ink4b increase during senescence as does association of this kinase inhibitor with cyclinD/cdk4 complexes. However, p16ink4a, p53 and p19ARF expression also increase during senescence. Genetic analysis shows that TGFbeta1 null and dominant negative TbetaBRII expressing v-rasHa keratinocytes resist the G1 growth arrest and do not senescence. This resistance is associated with low expression of p15ink4b and p16ink4a, constitutive Rb phosphorylation and high levels of cdk4 and cdk2 kinase activity. In contrast, inactivation of TGFbetabeta1 secretion or response does not block the induction of p53 and p19ARF, but the level of p21waf1, a p53 target gene, is reduced in cyclin D/cdk4 and cyclin E/cdk2 complexes. Thus, although multiple senescence pathways are activated in response to a ras oncogene, inactivation of TGFbeta1 secretion or response is sufficient to block the senescence program. Since v-rasHa transduced TGFbeta1-/- keratinocytes form squamous cell carcinomas following skin grafting, these results suggest that in mouse keratinocytes, defects in TGFbeta1 signaling accelerate malignant progression by overcoming oncogene induced replicative senescence.

A mechanism of suppression of TGF-beta/SMAD signaling by NF-kappa B/RelA.

A number of pathogenic and proinflammatory stimuli, and the transforming growth factor-beta (TGF-beta) exert opposing activities in cellular and immune responses. Here we show that the RelA subunit of nuclear factor kappaB (NF-kappaB/RelA) is necessary for the inhibition of TGF-beta-induced phosphorylation, nuclear translocation, and DNA binding of SMAD signaling complexes by tumor necrosis factor-alpha (TNF-alpha). The antagonism is mediated through up-regulation of Smad7 synthesis and induction of stable associations between ligand-activated TGF-beta receptors and inhibitory Smad7. Down-regulation of endogenous Smad7 by expression of antisense mRNA releases TGF-beta/SMAD-induced transcriptional responses from suppression by cytokine-activated NF-kappaB/RelA. Following stimulation with bacterial lipopolysaccharide (LPS), or the proinflammatory cytokines TNF-alpha and interleukin-1beta (IL-1beta, NF-kappaB/RelA induces Smad7 synthesis through activation of Smad7 gene transcription. These results suggest a mechanism of suppression of TGF-beta/SMAD signaling by opposing stimuli mediated through the activation of inhibitory Smad7 by NF-kappaB/RelA.

Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta.

Smad7 is an inducible intracellular inhibitor of transforming growth factor-beta (TGF-beta) signaling that is regulated by diverse stimuli including members of the TGF-beta superfamily. To define the molecular mechanisms of negative control of TGF-beta signaling, we have isolated the human SMAD7 gene and characterized its promoter region. A -303 to +672 SMAD7 region contained a palindromic GTCTAGAC Smad binding element (SBE) between nucleotides -179 and -172 that was necessary for the induction of a Smad7 promoter luciferase reporter gene by TGF-beta. Electrophoretic mobility shift assays using oligonucleotide probes demonstrated that TGF-beta rapidly induced the binding of an endogenous SBE-binding complex (SBC) containing Smad2, Smad3, and Smad4. Transfection assays in mouse embryonic fibroblasts (MEFs), with targeted deletions of either Smad2 or Smad3, and the Smad4-deficient cell line MD-MBA-468 revealed that both Smad3 and Smad4, but not Smad2, were absolutely required for induction of the Smad7 promoter reporter gene by TGF-beta. Furthermore, the TGF-beta-inducible SBE-binding complex was diminished in Smad2-deficient MEFs when compared with wild type MEFs and not detectable in Smad3-deficient MEFs and MD-MBA-468 cells. Taken together, our data demonstrate that TGF-beta induces transcription of the human SMAD7 gene through activation of Smad3 and Smad4 transcription factor binding to its proximal promoter.