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.

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.

Transforming growth factor-beta1 is a new form of tumor suppressor with true haploid insufficiency.

Components of the transforming growth factor-beta (TGF-beta) signal pathway function as classic tumor suppressors, but the role of the TGF-betas themselves is less clear. Here we show that mice heterozygous for deletion of the TGF-beta1 gene express only 10-30% of wild-type TGF-beta1 protein levels. Although grossly normal, these mice have a subtly altered proliferative phenotype, with increased cell turnover in the liver and lung. Treatment of these mice with chemical carcinogens resulted in enhanced tumorigenesis when compared with wild-type littermates. However, tumors in the heterozygous mice did not lose the remaining wild-type TGF-beta1 allele, indicating that the TGF-beta1 ligand is a new form of tumor suppressor that shows true haploid insufficiency in its ability to protect against tumorigenesis.

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.

The human beta 2 integrin CD18 promoter consists of two inverted Ets cis elements.

To define the minimal promoter responsible for expression of CD18 in myeloid and lymphoid cells, we generated 5' and 3' deletion constructs of a segment extending 785 bp upstream and 19 bp downstream of a major transcription start site and determined their effects on driving expression of the luciferase reporter gene in transfected hematopoietic cell lines. A region extending from nucleotides (nt) -302 to +19 was sufficient for cell-restricted and phorbol ester-inducible expression. DNase I footprinting of this region revealed two adjacent protected segments extending from nt -81 to -68 (box A) and -55 to -41 (box B). When a construct of 47 nt in length containing box A and box B and lacking other 3' or 5' elements was cloned into a promoterless vector, it conferred tissue-specific and phorbol ester-inducible expression. Gel retardation revealed that the protein components of two major protein-DNA complexes that form on both box A and box B and are required for transcriptional activation are members of the Ets oncoprotein family; one is related to the GA-binding protein (GABP), and the other is related to PU.1/Spi-1. The minimal CD18 promoter, lacking TATA, CAAT, and initiator elements and consisting primarily of Ets repeats, may exemplify an emerging class of promoters with which the concerted binding of Ets factors is necessary and sufficient to mediate transcriptional activation through direct recruitment of the basal transcription machinery.

Transgenic mice overexpressing a dominant-negative mutant type II transforming growth factor beta receptor show enhanced tumorigenesis in the mammary gland and lung in response to the carcinogen 7,12-dimethylbenz-[a]-anthracene.

To test the hypothesis that the transforming growth factor-beta (TGF-beta) system has tumor suppressor activity in the mammary gland, we have generated transgenic mice overexpressing a dominant-negative mutant form of the type II TGF-beta receptor, under the control of the mouse mammary tumor virus-long terminal repeat. High-level expression of the transgene was observed in the mammary and salivary glands, with lower expression in the lung, spleen, and testis. Older nulliparous transgenic mice (9-17 months) showed a marked increase in the incidence and degree of lobulo-alveolar side-branching in the mammary glands when compared to wild-type littermates (24.8% of glands examined histologically versus 14.4%; P = 0.004), suggesting a role for endogenous TGF-betas in regulating development or maintenance of mammary alveoli. Spontaneous tumorigenesis was unchanged in the transgenic mice. However, following initiation with the carcinogen 7,12-dimethylbenz[a]anthracene, the transgenic group showed a significant increase in the incidence and multiplicity of mammary tumors when compared with wild-type littermates (40% incidence in transgenic mice versus 22% for wild-type, with 4 of 25 transgenics developing multiple mammary tumors versus 0 of 27 wild-type; P = 0.03). An early increase in the incidence of lung tumors was also observed in transgenic mice, but no difference between genotype groups was seen in the incidence of tumors in tissues in which the transgene is not expressed. The data show that the endogenous TGF-beta system has tumor suppressor activity in the mammary gland and lung.

Loss of responsiveness to transforming growth factor beta induces malignant transformation of nontumorigenic rat prostate epithelial cells.

Transforming growth factor (TGF)-betas are multifunctional growth factors, the properties of which include the potent inhibition of epithelial cell growth. Expression patterns of TGF-betas and TGF-beta receptors in the normal prostate indicate that these growth regulators play key roles in prostatic development and proliferative homeostasis. Importantly, TGF-beta receptor levels are frequently diminished in malignant human prostate tissue. To test the hypothesis that loss of TGF-beta responsiveness is causally involved in the tumorigenic process, we have used retroviral transduction to introduce a dominant-negative mutant type II TGF-beta receptor (DNR) into the premalignant rat prostatic epithelial cell line, NRP-152. High-level expression of the DNR abolished the ability of TGF-beta to inhibit cell growth, to promote cell differentiation, and to induce apoptosis, and it partially blocked the induction of extracellular matrix gene expression. When injected into nude mice, NRP-152-DNR cells formed carcinomas at 13 of 34 sites, compared with 0 of 30 sites for parental and control cells (P = 0.0001). We conclude that the type II TGF-beta receptor is an important tumor suppressor in the prostate, and furthermore, that loss of TGF-beta responsiveness can contribute early in the tumorigenic process by causing the malignant transformation of preneoplastic cells.

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.

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.

The syndrome of lung hemorrhage and nephritis is usually an ANCA-associated condition.

BACKGROUND: In the absence of evidence of arteritis or Wegener's granulomatosis, the syndrome of lung hemorrhage and nephritis has been commonly associated with anti-glomerular basement membrane (GBM) antibodies. However, it has been increasingly recognized that many cases are associated with antineutrophil cytoplasmic antibodies (ANCAs). OBJECTIVE: To review available clinical and pathologic findings to determine the diseases accounting for lung hemorrhage and nephritis. METHODS: We studied the records of 750 patients from whom serum samples were sent to our laboratory for anti-GBM antibody assays between 1981 and 1993 and found 88 patients with evidence of lung hemorrhage and nephritis. Serum samples were retested, using current methods, for anti-GBM antibodies (against noncollagenous 1 domain of the alpha 3 chain of type IV collagen) and for antibodies to proteinase 3 and myeloperoxidase--the two types of ANCA of diagnostic value. RESULTS: Of 88 patients with evidence of lung hemorrhage and nephritis, 48 had ANCAs, six had anti-GBM antibodies, and seven had both. In 48 patients with ANCAs, the pathologic findings that accounted for the pulmonary renal syndrome were pauci-immune necrotizing and crescentic glomerulonephritis and pulmonary capillaritis. Only eight had convincing evidence (during life) of Wegener's granulomatosis and only one other had documented arteritis. In 27 patients without ANCAs or anti-GBM antibodies, a variety of unrelated renal and pulmonary diseases were found. CONCLUSIONS: The largest group of patients who present with the syndrome of lung hemorrhage and nephritis have ANCAs and not anti-GMB antibodies. Appropriate tests for antibodies to proteinase 3, antibodies to myeloperoxidase, and anti-GBM antibodies provide reliable guides for making a diagnosis in patients with this pulmonary renal syndrome.

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.

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.

Lessons from TGF-beta transgenic mice.

Aberrant expression of TGF-beta and/or receptor/signaling function is present in a wide variety of disease processes. Overexpression of TGF-beta isoforms in transgenic mice using tissue-specific promoters has provided model systems to study the effects of increased activity of TGF-beta in the intact organism. We will review the pertinent features of some of these models, and discuss new insights provided by these studies into regulation and role of TGF-beta in health and disease.

TGF-beta knockout and dominant-negative receptor transgenic mice.

Use of homologous recombination and transgenic technologies have provided mouse models to study the physiological roles of the three mammalian TGF-beta isoforms, and their regulation in the context of the intact animal. Mice harboring null mutations for TGF-beta isoforms demonstrate that each exerts discrete nonoverlapping functions during development. TGF-beta1 null mice reveal a crucial role for this cytokine in modulation of the immune system, with evidence for altered development, activation and function of various immune cell populations. New approaches to tissue- and cell-restricted disruption of TGF-beta signaling pathways in transgenic mice carrying dominant-negative mutant TGF-beta receptors will be discussed.

Biology of TGF-beta in knockout and transgenic mouse models.

This paper reviews the basic biology and biochemistry of the TGF-beta isoforms including their unique serine-threonine receptors and signaling intermediates. Dysregulation of TGF-beta expression and/or receptor/signaling function have been implicated in a wide variety of pathologies. We will discuss mechanisms underlying some of these disease processes as gained from study of transgenic mice in which expression of TGF-beta 1 has either been lost by targeted deletion of its gene, is overexpressed in a tissue-specific manner, or blocked by its latency associated peptide.

Transforming growth factor-beta in renal disease.

An extensive number of animal and clinical studies indicate that transforming growth factor-beta (TGF-beta s) play an important role in inflammatory and fibrotic diseases, including renal fibrosis. Recent mouse models harboring genetically engineered alterations in TGF-beta pathways reveal complicated mechanisms of regulation of TGF-beta activity in vivo. The purpose of this review is to present recent advances relevant to our understanding of the TGF-beta-signaling system in renal physiology and pathophysiology.

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.

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.

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.