Smoothed Lexis Diagrams With Applications to Lung and Breast Cancer Trends in Taiwan

<div><p>Cancer surveillance research often begins with a rate matrix, also called a Lexis diagram, of cancer incidence derived from cancer registry and census data. Lexis diagrams with 3- or 5-year intervals for age group and for calendar year of diagnosis are often considered. This simple smoothing approach suffers from a significant limitation; important details useful in studying time trends may be lost in the averaging process involved in generating a summary rate. This article constructs a smoothed Lexis diagram and indicates its use in cancer surveillance research. Specifically, we use a Poisson model to describe the relationship between the number of new cases, the number of people at risk, and a smoothly varying incidence rate for the study of the incidence rate function. Based on the Poisson model, we use the standard Lexis diagram to introduce priors through the coefficients of Bernstein polynomials and propose a Bayesian approach to construct a smoothed Lexis diagram for the study of the effects of age, period, and cohort on incidence rates in terms of straightforward graphical displays. These include the age-specific rates by year of birth, age-specific rates by year of diagnosis, year-specific rates by age of diagnosis, and cohort-specific rates by age of diagnosis. We illustrate our approach by studying the trends in lung and breast cancer incidence in Taiwan. We find that for nearly every age group the incidence rates for lung adenocarcinoma and female invasive breast cancer increased rapidly in the past two decades and those for male lung squamous cell carcinoma started to decrease, which is consistent with the decline in the male smoking rate that began in 1985. Since the analyses indicate strong age, period, and cohort effects, it seems that both lung cancer and breast cancer will become more important public health problems in Taiwan. Supplementary materials for this article are available online.</p></div

Additional file 1: Table S1. of Cardiometabolic disorder reduces survival prospects more than suboptimal body mass index irrespective of age or gender: a longitudinal study of 377,929 adults in Taiwan

All-cause mortality risk for subjects overall and those who survived at least 1, 2, and 3 years after entry. Figure S1. Adjusted mortality risk for different BMI, high blood pressure, hyperglycemia, and waist circumference for overall subjects, non-smokers, men, and women, stratified by age. BMI classification: underweight: <18.5 kg/m2, low normal: 18.5–21.9 kg/m2, normal: 22–23.9 kg/m2, overweight: 24–26.9 kg/m2, obese1: 27–29.9 kg/m2, obese2: ≥ 30 kg/m2. The hazards ratios shown in Figure S1 were derived from Cox proportional hazards models adjusted for gender, age, education level, smoking status, physical activity, and drinking status. Table S2. Mortality rate (per 10,000 person-years) by body mass index, age, and status of metabolic syndrome for the study subjects including previous heart disease and stroke (N = 390,941). Table S3. Mortality risk, prevalence, and population attributable burden of mortality for different BMI, high blood pressure, and hyperglycemia in overall subjects and people in different age groups, for the study subjects including previous heart disease and stroke (N = 390,941). (DOCX 135 kb

Disease-Related Growth Factor and Embryonic Signaling Pathways Modulate an Enhancer of <i>TCF21</i> Expression at the 6q23.2 Coronary Heart Disease Locus

<div><p>Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor <i>TCF21</i> gene. TCF21 (Capsulin/Pod1/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a <i>cis</i>-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1) element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered <i>TCF21</i> expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-β) and Wilms tumor 1 (WT1) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at <i>TCF21</i>.</p></div

Allele-specific transcriptional activity at rs12190287.

<p>(<b>a</b>) Transcriptional activity of rs12190287-C and G variants were determined in heterozygous primary human coronary artery smooth muscle (HCASM), rat aortic smooth muscle cells (RASM) and HEK, HepG2, and A7r5 cell lines. pLuc-MCS vector containing the putative enhancer region for each rs12190287 variant (C-Luc and G-Luc) was transfected for 24 hours and the ratio of firefly and <i>Renilla</i> luciferase activities were normalized to empty reporter (pLuc). *P<0.01 versus G-Luc for each condition. (<b>b</b>) Dual-luciferase assay of wildtype rs12190287 enhancer or mutants, DelT or T/A (as shown), transfected in A7r5 as described above. *P<0.001 versus G-Luc for each condition. (<b>c</b>) Electrophoretic mobility shift assays (EMSA) showing protein binding to [γ³²P]ATP-labeled rs12190287 C/G probes incubated with nuclear extract (NE) from various cell types, along with 100× excess C, G, or negative control (C, G, or Ctrl comp) unlabeled probe as competitor. Arrows and bar-headed lines represent specific and non-specific shifted complexes, respectively. (<b>d</b>) Dual-luciferase assay of rs12190287 C/G enhancer co-transfected with empty vector (Empty) or constitutively active protein kinase A (PKA) or mitogen-activated protein kinase (MEKK) in A7r5 using consensus CRE and AP-1 reporters as positive controls. *P<0.001 versus C-Luc+Empty or CRE-Luc+Veh or AP1-Luc+Veh where indicated. (<b>e</b>) EMSA of rs12190287 C/G with respective competition showing protein binding using control A7r5 or PMA treated A7r5 NE. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.</p

Predicted model of AP-1 dependent regulation of <i>TCF21</i> at rs12190287 and rs12524865.

<p>Individuals carrying risk alleles for rs12190287 or rs12524865 at 6q23.2 are expected to have increased <i>TCF21</i> expression upon stimulation of PDGFR-β by PDGF-BB in coronary artery smooth muscle cells, due to increased enrichment of active histone modifications (represented by closed and open diamonds) leading to an open chromatin conformation, allowing binding of an active AP-1 TF complex containing various combinations of c-Jun, JunD, and ATF3. WT1 functions as a transrepressor of this active complex at rs12190287, whereby WT1 may fine-tune the spatial and temporal activation of <i>TCF21</i> expression. Multiple kinases other than mitogen-activated kinase (MEKK) may be involved in the activation and recruitment of AP-1 complexes to these binding sites.</p

AP-1 regulation at rs12524865 and haplotype structure in East Asians.

<p>(<b>a</b>) Linkage disequilibrium (LD) plot from ∼2400 Metabochip genotyped East Asian samples from the TAICHI study (HALST cohort) with imputation from HapMap Phase II and III Han Chinese (CHB), showing eSNPs for <i>TCF21</i> and distinct haplotype blocks containing rs12524865 and rs12190287. White to black squares represent increasing r² values, also shown in blocks. (<b>b</b>) Dual-luciferase assay of rs12524865 C/A enhancer transfected in A7r5 and treated with adenylyl cyclase activator, forskolin (Fsk) or PKC activator, phorbol-12-myristate-13-acetate (PMA) for 4 hours. Relative luciferase activities measured after 24 hours. *P<0.01 versus C-Luc+Veh. (<b>c</b>) Total enrichment of c-Jun, JunB, JunD, and ATF3 at rs12524865 determined by chromatin immunoprecipitation (ChIP) in HCASMC treated with PDGF-BB or vehicle (Control) for 6 hours. *P<0.005 versus Control for each condition. (<b>d</b>) Total enrichment of histone modifications H3K4me1, H3K4me3, H3K27ac, and H3K27me1 at rs12524865 determined by ChIP in HCASMC treated with PDGF-BB. *P<0.001 versus Control for each condition. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.</p

WT1 regulation of rs12190287 <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>a</b>) Dual-luciferase assay of rs12190287 C/G enhancer transfected with human WT1-B (−KTS), WT1-D (+KTS) expression constructs in A7r5 cells, measured after 24 hours. *P<0.01 versus C-Luc or G-Luc+Empty. (<b>b</b>) Dual-luciferase assay of rs12190287 C/G enhancer co-transfected with human c-JUN and WT1-B or WT1-D expression constructs in A7r5 cells. AP1-Luc reporter was used as a positive control. *P<0.05 versus C-Luc or G-Luc+cJun. (<b>c</b>) Dual-luciferase assay of rs12190287 C/G enhancer transfected in heterozygous HCASMC with siRNA against <i>WT1</i> (−/+KTS) compared to negative control (Neg si). *P<0.05 versus C-Luc or G-luc+Neg si. (<b>d</b>) TaqMan based qRT-PCR results showing relative human <i>WT1</i> mRNA expression levels in heterozygous HCASMC treated with TGF-β1 or PDGF-BB for the indicated times. (<b>e</b>) Total enrichment of WT1 at rs12190287 enhancer, <i>FOSB</i> or <i>MYOG</i> promoter regions determined by chromatin immunoprecipitation (ChIP) in heterozygous HCASMC treated with PDGF-BB for 6 hrs. Values represent fold change relative to enrichment with IgG control. *P<0.01 versus Control WT1. (<b>f</b>) Allele-specific enrichment of WT1 at rs12190287 determined by HaploChIP in heterozygous HCASMC treated with PDGF-BB, shown as normalized allelic-ratio C/G. *P<0.0005 versus Control WT1. Values are mean ± SD from triplicates. Similar results were observed from three independent experiments.</p

<i>In silico</i> allele-specific transcription factor binding to rs12524865.

<p>Predicted transcription factor binding site searches were performed using five programs: TRANSFAC, PROMO, MatInspector, TFSearch, and JASPAR. The SNP is shown in boldface within the predicted binding sequence. Core binding sequence shown in capital letters for TRANSFAC and MatInspector generated sequences. The scores represent matrix sequence similarity for each program. A minimum threshold of 85.0 was used for each program, and lowered to 75.0 for allelic comparison if necessary.</p

PDGFRβ is upstream of <i>TCF21</i> at rs12190287 in HCASMC.

<p>(<b>a</b>) TaqMan based qRT-PCR results showing relative mRNA expression levels of total <i>TCF21</i> in heterozygous HCASMC treated with TGF-β1, PDGF-BB or PMA for indicated times. Expression levels were normalized to 18S and expressed as fold change from 0 h. *P<0.0001 versus 0 h. (<b>b</b>) TaqMan qRT-PCR expression results of c-<i>JUN</i>, <i>JUND</i>, and <i>ATF3</i> in heterozygous HCASMC treated TGF-β1 or PDGF-BB for the indicated times. (<b>c</b>) Allele-specific TaqMan qRT-PCR based expression of <i>TCF21</i> at rs12190287 shown as normalized allelic ratio C/G in heterozygous HCASMC treated with either TGF-β1 or PDGF-BB for the indicated times. *P<0.01 versus 0 h. Values are mean ± SD of triplicates. Similar results were observed from three independent experiments.</p

<i>In silico</i> allele-specific transcription factor binding to rs12190287.

<p>Predicted transcription factor binding site searches were performed using five programs: TRANSFAC, PROMO, MatInspector, TFSearch, and JASPAR. The SNP is shown in boldface within the predicted binding sequence. Core binding sequence shown in capital letters for TRANSFAC and MatInspector generated sequences. The scores represent matrix sequence similarity for each program. A minimum threshold of 85.0 was used for each program, and lowered to 75.0 for allelic comparison if necessary.</p