Approved COPDGene® Ancillary Studies
Approved COPDGene® Ancillary Studies
Proteomics and Metabolomics in COPDGene®
PI: Russell Bowler, MD, PhD
Active: Yes
Funding: Funded
Sites: National Jewish Health, University of Iowa, L.A. Biomedical Research Institute,
Baylor College of Medicine, open to additional centers
The purpose of this project is to identify plasma metabolic and proteomic profiles that are
associated with high resolution CT-defined (HRCT) emphysema and airways disease. The project
will also determine genetic control of candidate biomarkers and replicate findings in independent
populations. As of October 28, 2009 there are 588 P100 tubes (fresh-frozen aliquots with protease
inhibitor) in the study database. A pooled plasma sample from 200 subjects has been created for use
in standardization assays. Preliminary proteomic analysis has begun and the investigator has
partnered with Steven Kelsen, MD for a GO proteomics grant.
Plasma Proteomics in Chronic Obstructive Pulmonary Disease (COPD)
PI: Steven Kelsen, MD
Active: Yes
Funding: Funded
Sites: Temple University,
Proteomic approaches afford considerable power in identifying novel or differentially expressed
proteins and their downstream signaling pathways which are pathogenetically important in diseases
like COPD 1-5. Such approaches have shown that cigarette smoke induces a compensatory
response termed the unfolded protein response (UPR) in the human lung in response to endoplasmic
reticulum (ER) stress, a condition in which the capacity to fold, modify and transport mature
secretory and membrane proteins cannot keep pace with the rate of protein synthesis 3. In other
cells and tissues, the UPR exerts important anti-oxidant and anti-inflammatory effects that prevent
cell death and tissue destruction. In the setting of cigarette smoke exposure, it is possible that the
UPR protects lung cells against oxidant stress and the development of COPD. The overall goal of
this project is to identify plasma biomarkers which define disease susceptibility and severity in
chronic smokers with and without COPD enrolled in the COPDGene® study. The project will
identify candidate plasma markers of disease susceptibility and severity in an observational group of
smoking subjects at risk for COPD (GOLD 0) and subjects with severe COPD (GOLD 4). The team
will also examine the utility of candidate plasma biomarkers in a separate, expanded cohort of
subjects with a wide range of COPD severity, i.e., GOLD stages 0, 1, 2, 3 and 4.
Thoracic Aorta Calcification and Coronary Artery Calcification in the COPDGene® Study
PI: Matthew J. Budoff, MD
Active: No
Funding: Submitted to NHLBI
Sites: Utilizes CT Scans
The project will collect throacic aortic and coronary aortic calcium imaging data to provide novel
insights into the interaction between phenotype/genotype expression of COPD and subclinical
atherosclerosis. In addition, prospective follow-up of these patients will establish the value of
thoracic and coronary artery calcification in prediction of coronary heart disease in patients with
COPD. This will provide an opportunity to assess the complex nature of interactions between the
pulmonary and cardiovascular systems at subclinical levels before the occurrence of clinically
apparent cardiovascular disease.
High-throughput Study of DNA Methylation in Smokers With and Without COPD
PI: Dawn L. DeMeo, MD, MPH
Active: No
Funding: Submitted to NHLBI
Sites: Will be done in Genetic Analysis Core
This project will use state of the art high-throughput epigenetic methods to investigate features of
COPD susceptibility and COPD subtypes. Whole-genome methylation patterns, integrated with
genome-wide association analysis, will provide the most comprehensive investigation of geneticepigenetic
influences on COPD susceptibility. The project aims include extracting DNA from
buccal brush DNA, performing an Ilumina Infinum Genome-wide methylation assay for buccal
brush DNA and peripheral blood DNA, doing a subsequent analysis of methylation both within and
between case and controls groups, and finally analyzing the methylation patterns of COPD cases
with airway-predominant and COPD cases emphysema-predominant.
Molar Mass Measurements during Quiet Breathing
PI: Robert L. Jensen, PhD
Active: Yes
Funding:
Sites: Brigham and Women's Hospital, University of California, San Diego, open to
additional centers
This project uses the current ndd Spirometer to measure the molar mass of airflow during quiet
breathing for approximately two minutes. These parameters have been shown to distinguish
"normal" from "obstructive" patterns and can be used to correctly categorize patients into
obstructive patterns about 80% of the time. The project will be able to further correlate molar mass
measurements to quantitative high resolution CT in order to demonstrate key associations with both
degree and location of altered lung anatomy in the presence of COPD.
Genetic Associations with Body Composition and Musculoskeletal Disease in COPD
PI: Elizabeth A. Regan, MD, PhD
Active: No
Funding: Submitted to NHLBI
Sites: All interested study centers
This project will analyze the relationship of muscle wasting and low bone density in individuals
with COPD with other aspects of the disease, such as exacerbations, chronic inflammation, poor
exercise tolerance and radiographic phenotypes. Specifically the project proposes to obtain whole
body DXA scans for muscle mass, fat mass and bone density in a subset of the cohort to compare to
measures of bone density, muscle volume and fat mass on the HRCT done for COPDGene®.
Supplemental questionnaires will collect more detailed information about musculoskeletal
symptoms and diagnoses. Using that data we will assess the impact of osteoporosis and reduced
muscle mass on COPD subjects and identify genetic markers associated with increased risk of
musculoskeletal disease, decreased muscle mass and low bone density.
Ancillary 129Xe Studies for COPDGene®
PI: Samuel Patz, PhD
Active: Yes
Funding: Submitted to NHLBI
Sites: Brigham and Women's Hospital
COPDGene® subjects at BWH will be invited to participate in this ancillary study. Using hyperpolarized
129Xe, the project proposes establishing a baseline statistical distribution of pulmonary
surface area to volume ratio, septal thickness, and capillary transit times. 129Xe measures of disease
severity in GOLD Stage 1-4 subjects are postulated to be more highly correlated with physical
disabilities associated with their pulmonary disease than traditional tests of pulmonary function. The
project will extend 129Xe measurements from whole lung to regional measurements. They will
determine the degree to which the heterogeneity of regional 129Xe measurements of pulmonary
function in a single individual correlates with physical manifestations of disease severity. They
hypothesize that measures of spatial heterogeneity will correlate highly with physical disability. The
study has begun enrollment and a manuscript is in development.
Integrative Genomics of the Genetics of Early Interstitial Lung Disease
PI: Ivan Rosas, MD
Active: Yes
Funding: Submitted to NHLBI
Sites: Utilize CT scans and GWAS data
Idiopathic Pulmonary Fibrosis (IPF), the most common human idiopathic interstitial lung disease
(ILD), is a chronic progressive disorder with an estimated survival of 20-50% at 3 years. The early
molecular events that lead to the development of IPF are poorly understood. We hypothesize that
common genetic variation in a small subset of genes confers susceptibility to the development of
early ILD in smokers. The aims of the study are to 1) Determine the percentage of subjects with
early ILD detected using chest CT in smokers of the COPDGene® study. 2) Determine demographic
(age, gender, ethnicity and exposures) and clinical characteristics (medical history, symptoms, PFT)
of smokers with early ILD. 3) Identify a focused set of genes deregulated both in peripheral blood
of smokers with early ILD and the lung of subjects affected with familial pulmonary fibrosis and
early ILD. To date the study team has reviewed 2,500 CT scans and identified preclinical ILD
changes in 8% of subjects.
Environmental Factors, Vitamin D and C-reactive Protein in COPD Exacerbation
PI: Susan Sama, ScD
Active: No
Funding: Submitted to NHLBI
Sites: Fallon Clinic
The proposed research will evaluate the effects of home, work and community exposures on the
exacerbation of COPD. The study population will include mild, moderate and severe cases of
COPD who are participating in the COPDGene® study and belong to the disease management group
(DMG) of a large multi-specialty group practice Health Maintenance Organization (HMO), the
Fallon Clinic and Fallon Community Health Plan. Using a case-crossover design, we will identify
short-term environmental triggers for exacerbation events. We hypothesize that recent home, work
and community exposures to dusts, irritant gases and fumes including agents known to precipitate
asthma exacerbations (molds and other respiratory irritants and sensitizers) will be associated with
COPD exacerbation. Data will be gathered by a detailed modular questionnaire on activities with
the potential to generate these exposures.
Interstitial Lung Disease GWAS R01
PI: David Schwartz, MD
Active: Yes
Funding: Funded
Sites: Utilize SNP data from GWAS
The goal of this proposal is to discover genes and gene variants that are central to the development
of fibrosing interstitial lung disease (ILD). Since both gene variants and the environment are known
to increase the risk of developing ILD, we seek to comprehensively identify the gene variants
associated with ILD by considering environmental exposures when studying the genetics of this
complex disease. The proposed project includes a genome-wide association study (GWAS) in 1600
patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP) and 2000
unaffected controls from the COPDGene® study for the first specific aim. They also propose to
perform a genome-wide association study (GWAS) in 1000 patients with asbestosis and 1000 nondiseased, asbestos-exposed controls. Finally, they will identify genetic markers and loci that are
unique to IPF/UIP vs. those that are unique to asbestosis. The COPDGene® population will be a
control population for this study. The study team expects to have the required number of cases by
early 2010 and genotyping completed by the Spring.
COPD CRN STATIN Trial
PI: George Washko, MD
Active: Yes
Funding: funded
Sites: Brigham and Women's Hospital
The study will recruit 200 COPD patients with at least moderate disease who are prone to
exacerbations. Patients will be randomized to one of four arms: simvastatin 20, 40 or 80 mgs, or
matched placebo, in one daily dose, for 6 months. Patients will have inflammatory biomarkers (CRP
and IL-6) as well as other cytokines and proteases (e.g., IL-10, TNFα, IL-17, MMP-9, among
others) measured at baseline prior to randomization and then at one, three, six, and seven months
post randomization.
Phase III Clinical Trial: Prospective Randomized Placebo-Controlled Trial of SimvaSTATin in the Prevention of COPD Exacerbations (STATCOPE)
PI: Gerard Criner, MD
Active: Yes
Funding: Funded
Sites: Univ. of Maryland at Baltimore, Univ. of Alabama at Birmingham, Birmingham
VAMC, Brigham and Women's Hospital, Fallon Clinic, National Jewish Health,
Denver VAMC, Denver Health, Harbor UCLA, Minneapolis VAMC, Health
Partners Research Foundation, Univ. of California, San Francisco, Ann Arbor
VAMC, Univ. of Michigan, Temple University, Univ. of Pittsburgh,
Pittsburgh VAMC
The purpose of the study is to determine the effect of daily administration of 40 mgms simvastatin
taken for at least 12 months (range 12‐36 months) on the frequency of exacerbations of chronic
obstructive lung disease (COPD) in patients with moderate to severe COPD who are prone to
exacerbations and do not have other indications for statin treatment. 1126 patients with at least
moderately severe COPD (postbronchodilator FEV1/FVC < 70%, postbronchodilator FEV1 < 80%
predicted, with or without chronic symptoms (i.e., cough, sputum production) who have an
increased risk for an exacerbation of COPD during the conduct of the trial. A companion
application to this submission is currently under review at the CIHR (Canadian Institute for Health
Research) Participants will be males and females 40‐80 yrs of age with cigarette consumption of
10 pack years or more. Participants may or may not be active smokers. To enrich the population for
patients who are more likely to have acute exacerbations, each participant must meet one or more of
the following 4 conditions:
1. Be using supplemental O2, 2. Received a course of systemic corticosteroids and/or antibiotics for
respiratory problems in the past year, 3. Visited an Emergency Department for a COPD
exacerbation within the past year, or 4. Be hospitalized for a COPD exacerbation within the past
year . Patients must also be willing to make return visits and be available by telephone for the
duration of study, be free of active coronary artery disease or other atherosclerotic vascular disease,
and be expected to have a life expectancy > 36 months.
Admixture mapping to identify areas of the genome associated with quantitative and qualitative COPD traits in African Americans
PI: Adam Friedlander, MD
Active: No
Funding:
Sites: Utilize CT scans and GWAS data
Although chronic obstructive pulmonary disease (COPD) has long been considered a disease of
non-Hispanic Caucasian men, recent epidemiologic data has demonstrated that mortality from
COPD is rising faster in African Americans when compared to whites. Although the exact reasons
for this changing demographic remain unclear, there is evolving evidence that African Americans
may be more susceptible to the damaging effects of tobacco smoke . Admixture mapping is a
technique that is useful in detecting genes that contribute to ethnic variation in disease risk. The
study hypothesizes that among African Americans there exist susceptibility markers for COPD and
COPD traits that are strongly associated with either European or African continental ancestry. The
aims of the study are to determine whether continental ancestry is a significant independent
predictor of the development of COPD and variability in COPD traits and to use admixture mapping
(MALD) to determine specific genetic loci responsible for associations between continental
ancestry and COPD traits.
Pharmacokinetics of statin therapy in COPDGene®
PI: Craig Hersh, MD
Active: Yes
Funding:
Sites: Utilize CT scans and GWAS data
Genetic variants may explain differences in responses to COPD treatment. Statins are a potential
new therapy for COPD, and pharmacogenetic influences on statin benefit and on statin toxicity have
been reported in cardiovascular studies. Statins are widely prescribed in patients with COPD for
coexisting hyperlipidemia, but not as a treatment for COPD. Therefore, statin therapy can be
examined in observational studies such as COPDGene®, as a complement to the COPD Clinical
Research Network's clinical trial of simvastatin. The study will analyze GWAS and candidate gene
genotyping data from COPDGene® subjects co-enrolled in the CCRN statin trial to identify genetic
predictors of effects of statin therapy in COPD and to analyze the GWAS and candidate gene
genotyping data in subjects already taking statin drugs to identify genetic predictors of response to
statin therapy, measured by exacerbations recorded during the COPDGene® telephone follow-up.
Hypoxemia and pulmonary vascular disease in COPDGene®
PI: Craig Hersh, MD
Active: Yes
Funding: Funded
Sites: Utilize CT scans and GWAS data
Hypoxemia and pulmonary hypertension are two inter-related complications of COPD, which lead
to increased morbidity and mortality. Hypoxemia can lead to an increase in pulmonary artery
pressures. Improvement in pulmonary artery pressure is one of the likely mechanisms of the benefit
of supplemental oxygen in COPD. The COPDGene® study presents a unique opportunity to
evaluate the epidemiologic predictors of hypoxemia and pulmonary hypertension and to extend
previous genetic association work, using the large cohort of well-characterized COPD patients and
control smokers. The study will examine the genetics of hypoxemia and pulmonary vascular
disease, determine clinical and radiographic predictors and monitor longitudinal effects of the
diseases.
A computational fluid dynamics model of inhalational injury and correlation to CT measures of emphysema and airway disease
PI: George Washko, MD
Active: Yes
Funding: Yes
Sites: Utilize CT scans and PFT data
It has be a recurring observation that for a given amount of tobacco smoke exposure, some subjects
are prone to the development of emphysema or airway disease. Using tobacco smoke as the
exposure model, the regional distribution of computed tomographic measures of both emphysema
and airway disease will be examined and correlated with the 3-Dimensional morphology of the
tracheobronchial tree in current and former smokers. A validated airflow/deposition model will be
applied to each subject's tracheobronchial tree to predict under simulated conditions of respiration
the degree and distribution of particle deposition in the airways and distal parenchyma. The
regional burden of airway and parenchymal disease will then be compared to the idealized particle
deposition pattern obtained under simulated conditions. The study team hopes to better understand
the influence of airway geometry on the distribution of emphysema and airway disease in smokers
and to investigate the type of particulate that may be responsible for the development of these
processes.
CT based measures of pulmonary vascular disease in subjects with COPD
PI: George Washko, MD
Active: Yes
Funding:
Sites: Utilize CT scans
Previous work suggests that CT based measures of the pulmonary vasculature correlate with both a
subject's burden of emphysema and invasive right heart catheterization based measures of
pulmonary vascular pressure and resistance. The study team has found that the CT measure of the
pulmonary vasculature termed cross sectional area, correlates with peripheral vascular disease
independent of a subject's burden of emphysema. The team will develop and implement an
automated tool for the quantification of pulmonary vascular disease in the COPDGene® Study
cohort. This measure could then be used for correlative investigation with other ancillary studies as
well as more focused investigation into secondary pulmonary vascular disease in subjects with
COPD.
Genetic Meta-Analysis and Predictive Modeling of COPD Susceptibility and COPDRelated Traits
PI: Peter Castaldi, PhD
Active: No
Funding:
Sites: Utilize GWAS data
Large-scale genome mapping efforts and advances in genotyping technology have provided the
necessary infrastructure for genome-wide efforts to identify genetic loci associated with common,
low-penetrance genetic disorders. This research has the potential for high public health impact,
since disorders such as diabetes, coronary artery disease, and COPD are highly prevalent; and the
identification of disease-causing genes could provide new therapeutic targets and improved
diagnostic tools. The task of identifying genes associated with these disorders has proven to be quite
difficult, and the translational potential of genome-wide association (GWA) studies is as yet
unrealized. The study will use state-of-the-art methods of genetic evidence synthesis and machine
learning to build predictive models for COPD and COPD-related traits that specifically include
gene-by-smoking and gene-gene interactions. These methods will be applied to a combined data
sample of 4 large COPD GWA studies (COPDGene®, NETT-NAS, ECLIPSE, GSK Norway). This
well-phenotyped study sample of individual subject level-data from over 16,000 subjects will be the
largest COPD case-control GWA sample assembled to date.
Copy Number Variation in COPD and COPD related phenotypes
PI: Emily Wan, MD
Active: Yes
Funding:
Sites: Utilize GWAS data
Structural variations, including copy number variations, influence the development of COPD and
various COPD-related phenotypes, including emphysema severity and distribution, airway disease
severity and distribution, airflow obstruction severity, and exacerbation frequency. The aims of the
study are to detect copy number variants in COPD cases and controls using data available from the
Illumina Omni-1 chip used for genome-wide SNP genotyping and to determine a copy number
association with disease status and COPD-related phenotypes by performing association studies
between well characterized CNV regions and case-control status as well as COPD-related
phenotypes such as emphysema index and FEV1% predicted.
Mitochondrial polymorphisms in COPD
PI: Emily Wan, MD
Active: Yes
Funding:
Sites: Utilize GWAS data
Despite playing a substantial role in forensics1 and evolutionary biology2, mitochondrial
genetic variants in complex diseases have remained largely unexplored. Rare syndromes attributed
to mitochondrial DNA mutations, such as Leber's hereditary optic neuropathy3, are analogous to the
variants of large effect sizes responsible for classical Mendelian disorders of the autosomal genome.
Studies investigating the role of mitochondrial variants in complex diseases, where the effect sizes
may be considerably more modest, have been limited.
The 16.6 kb mitochondrial genome is distinct in nearly every capacity from the nuclear
genome - the chromosome is circular, contains no introns, does not undergo recombination, and
demonstrates uniparental (maternal) inheritance4. In addition to their role in energy metabolism,
mitochondria play critical roles in the generation of reactive oxygen species5, 6, apoptosis7, 8, and
aging9 - processes of interest in the development of COPD. A preliminary analysis combining data
from three independent COPD cohorts (the National Emphysema Treatment Trial, the Norway-
Bergen cohort, and the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints
trial) revealed significant associations between five common mtSNPs and COPD casecontrol
status.
In a separate analysis, preliminary epidemiological data collected on a subset of subjects in
COPDGene with severe (FEV1% <40% predicted), early onset (age <53 years) COPD is suggestive
of an enhanced role for maternal factors. In an analysis conducted by our group, subjects with
EOCOPD reported increased rates of maternal COPD and emphysema when compared to non-early
onset COPD subjects. There were no significant associations with paternal lung disease.
Analysis of Sex Chromosome Variants
PI: Emily Wan, MD
Active: Yes
Funding:
Sites: Utilize GWAS data
Although the absolute number of women with COPD remains less than that of men, the death rate
from COPD and COPD-related causes in women increased substantially between 1971-2000, with
the absolute number of female deaths exceeding the number of male deaths in 20001. On average,
women with COPD have lower BMI2, increased dyspnea2-4, and increased rates of depression2. For
equivalent levels of spirometric impairment, women have a proportionally smaller decrease in
exercise capacity but overall lower maximal wattage on exercise testing2, 5, 6 and less emphysema2, 7 than males.
Though increasing appreciation for the clinical differences between the genders in COPD
has grown substantially in the last decade, advances into the genetic causes of such differences
remain largely unexplored. Increasing evidence supports the theory that women may be more
sensitive to the deleterious effects of cigarette smoke as women with COPD tend to be younger and
have less total smoking exposure than their male counterparts2, 5, 8. In a cohort of subjects with
severe, early-onset COPD not due to alpha-1 antitrypsin deficiency, a striking female predominance
was noted9. In addition, female first degree relatives of the probands who smoked had significantly
lower FEV1/FVC and a greater risk of profoundly reduced FEV1 than males10.
In a preliminary analysis on a subset of subjects recruited from the first 2500 subjects in
COPDGene with severe (FEV1<40%), early-onset (age <53 years at enrollment) COPD, a
significantly higher proportion of females (71%) was again noted. Consistent with previous reports,
these subjects had significantly fewer pack-years of smoking and less emphysema than COPD
controls.
Evaluating Deformable Image Registration Derived Imaging Biomarkers using the COPDGene® Dataset
PI: Thomas Guerrero, MD
Active: Yes
Funding: Submitted to NHLBI
Sites: University of California, San Diego
Two predominant disease manifestations are apparent on CT imaging, emphysema and small airway
disease. The research proposed in this study aligns with the research directions of the National
Heart Lung and Blood Institute (NHLBI) to further characterize and phenotype COPD. Both
COPDgene® and the proposed SPIROMICS studies will establish repositories of CT image data
with the goal of analyzing these CT images to obtain quantitative measures that may indicate the
presence and/or severity of COPD. In these studies, expiratory breath-hold CT (eBH-CT) and
inspiratory breath-hold CT (iBH-CT) imaging of the lung is performed. Each are evaluated
independently and there is no provision for comparing follow-up imaging. The study team has
linked eBH-CT and iBH-CT images in rodent models and in patients who received thoracic
irradiation to correlate the anatomic findings and to extract new physiological information such as
regionalcompliance and specific ventilation using deformable image registration (DIR) The method
for extracting ventilation images from CT using DIR is being tested in a National Cancer Institute
(NCI) funded study (R21CA128230). The ancillary study will further develop and validate these
methods to improve phenotyping of COPD, by detecting air-trapping, small airway disease,
estimating emphysema, and evaluating disease progression.
CT based assessment of airway remodeling using mural attenuation and "Power"
PI: Raul San Jose Estepar, MD
Active: Yes
Funding: Submitted to NHLBI
Sites: Utilizes CT scans
CT measures of airway wall attenuation can be used to predict lung function in subjects with COPD
and this measure can provide complimentary information to standard measures of WA% in defining
objective disease phenotypes. The study proposes to perform measures of airway wall attenuation
on the CT scans of subjects enrolled in the COPDGene® Study and examine the relationship of
these measures to lung function and additional information such as subject age, race, gender,
symptoms, functional capacity (as assessed by 6 MWD), etc. The team will examine the utility of a
much more experimental measure of mural remodeling termed "Power" in the COPDGene® cohort
and examine its relationship to clinical and functional measures of disease.
Diaphragm Remodeling and COPD Severity
PI: Aladin Boriek, PhD
Active:
Funding: Submitted to NHLBI
Sites: Utilizes CT scans
Diaphragm muscle dysfunction is widely thought to contribute to morbidity and mortality in COPD.
COPD is a multi-systemic and progressive obstructive airway disease that affects skeletal muscles
including the diaphragm. Several determinants of diaphragm function ultimately may lead to
respiratory pump failure and therefore clinical interest is highly focused in understanding those
determinants.
The ability of the diaphragm to generate trans-diaphragmatic pressure depends on two important
mechanical properties: 1) diaphragmatic shape including curvature and thickness and 2)forcegenerating
capacity. COPD with hyperinflation of the lungs may have an adverse effect on
diaphragm curvature that compromises respiratory pump function separately from muscle
weakness/wasting. Indeed, lung volume reduction surgery itself alleviates this adverse effect.
However, there is little understanding of the relationship between COPD severity, degree of
hyperinflation, and diaphragm curvature.
We hypothesized that diaphragm remodeling would be correlated with COPD severity and
pulmonary function. Aim 1 - We would like to establish how the diaphragm principal and minor
curvatures are altered in relationship to COPD severity and degree of hyperinflation. Aim 2 - we
would like to establish a quantitative model of volume displacement by the diaphragm in severe
COPD and non-COPD patients. Aim 3 - we would like to generate a geometric and kinematic finite
element model of the relationship of diaphragm curvature, diaphragmatic tension, and
transdiaphramatic pressure from severe COPD and non-COPD patients.