Clinical and biomarker modifiers of vitamin D treatment response: the Multi-Ethnic Study of Atherosclerosis.

BACKGROUND: Different 25-hydroxyvitamin D [25(OH)D] thresholds for treatment with vitamin D supplementation have been suggested and are derived almost exclusively from observational studies. Whether other characteristics, including race/ethnicity, BMI, and estimated glomerular filtration rate (eGFR), should also influence the threshold for treatment is unknown. OBJECTIVES: The aim was to identify clinical and biomarker characteristics that modify the response to vitamin D supplementation. METHODS: A total of 666 older adults in the Multi-Ethnic Study of Atherosclerosis (MESA) were randomly assigned to 16 wk of oral vitamin D3 (2000 IU/d; n = 499) or placebo (n = 167). Primary outcomes were changes in serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations from baseline to 16 wk. RESULTS: Among 666 participants randomly assigned (mean age: 72 y; 53% female; 66% racial/ethnic minority), 611 (92%) completed the study. The mean (SD) change in PTH was -3 (16) pg/mL with vitamin D3 compared with 2 (18) pg/mL with placebo (estimated mean difference: -5; 95% CI: -8, -2 pg/mL). Within the vitamin D3 group, lower baseline 25-hydroxyvitamin D [25(OH)D] was associated with a larger decline in PTH in a nonlinear fashion. With baseline 25(OH)D ≥30 ng/mL as the reference, 25(OH)D <20 ng/mL was associated with a larger decline in PTH with vitamin D3 supplementation (-10; 95% CI: -15, -6 pg/mL), whereas 25(OH)D of 20-30 ng/mL was not (-2; 95% CI: -6, 1 pg/mL). A segmented threshold model identified a baseline 25(OH)D concentration of 21 (95% CI: 13, 31) ng/mL as an inflection point for difference in change in PTH. Race/ethnicity, BMI, and eGFR did not modify vitamin D treatment response. There was no significant change in 1,25(OH)2D in either treatment group. CONCLUSIONS: Of characteristics most commonly associated with vitamin D metabolism, only baseline 25(OH)D <20 ng/mL modified the PTH response to vitamin D supplementation, providing support from a clinical trial to use this threshold to define insufficiency. This trial was registered at clinicaltrials.gov as NCT02925195.

Rationale and design of the Kidney Precision Medicine Project.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are common, heterogeneous, and morbid diseases. Mechanistic characterization of CKD and AKI in patients may facilitate a precision-medicine approach to prevention, diagnosis, and treatment. The Kidney Precision Medicine Project aims to ethically and safely obtain kidney biopsies from participants with CKD or AKI, create a reference kidney atlas, and characterize disease subgroups to stratify patients based on molecular features of disease, clinical characteristics, and associated outcomes. An additional aim is to identify critical cells, pathways, and targets for novel therapies and preventive strategies. This project is a multicenter prospective cohort study of adults with CKD or AKI who undergo a protocol kidney biopsy for research purposes. This investigation focuses on kidney diseases that are most prevalent and therefore substantially burden the public health, including CKD attributed to diabetes or hypertension and AKI attributed to ischemic and toxic injuries. Reference kidney tissues (for example, living-donor kidney biopsies) will also be evaluated. Traditional and digital pathology will be combined with transcriptomic, proteomic, and metabolomic analysis of the kidney tissue as well as deep clinical phenotyping for supervised and unsupervised subgroup analysis and systems biology analysis. Participants will be followed prospectively for 10 years to ascertain clinical outcomes. Cell types, locations, and functions will be characterized in health and disease in an open, searchable, online kidney tissue atlas. All data from the Kidney Precision Medicine Project will be made readily available for broad use by scientists, clinicians, and patients.

The Multi-Ethnic Study of Atherosclerosis individual response to vitamin D trial: Building a randomized clinical trial into an observational cohort study.

The INdividual response to VITamin D (INVITe) trial was a randomized, placebo-controlled, parallel group trial of vitamin D3 supplementation (2000 IU daily) designed to determine clinical and genetic characteristics that modify the response to vitamin D supplementation. To enhance internal and external validity and reduce cost, the INVITe trial was nested within the Multi-Ethnic Study of Atherosclerosis (MESA), an ongoing prospective observational cohort study. The INVITe trial enrolled a community-based population of 666 racially and ethnically diverse participants from January 2017 to April 2019. This represents 30% of 2210 MESA participants approached for screening, and 96% of those found to be eligible. Barriers to enrollment included delayed initiation of the trial relative to scheduled MESA study visits, a lower number of available MESA participants than expected, and a high prevalence (18%) of high-dose vitamin D supplementation (>1000 IU daily, an exclusion criterion). The final study visit was attended by 611 participants (92%), and median adherence was 98%. Our experience suggests that integration of a randomized trial into an existing observational cohort study may leverage strengths of the source population and enhance enrollment, retention, and adherence, although with limited enrollment capacity. The INVITe trial will use rigorously-collected data to advance understanding of individual determinants of vitamin D response.

Air pollution and subclinical interstitial lung disease: the Multi-Ethnic Study of Atherosclerosis (MESA) air-lung study.

We studied whether ambient air pollution is associated with interstitial lung abnormalities (ILAs) and high attenuation areas (HAAs), which are qualitative and quantitative measurements of subclinical interstitial lung disease (ILD) on computed tomography (CT).We performed analyses of community-based dwellers enrolled in the Multi-Ethnic Study of Atherosclerosis (MESA) study. We used cohort-specific spatio-temporal models to estimate ambient pollution (fine particulate matter (PM2.5), nitrogen oxides (NOx), nitrogen dioxide (NO2) and ozone (O3)) at each home. A total of 5495 participants underwent serial assessment of HAAs by cardiac CT; 2671 participants were assessed for ILAs using full lung CT at the 10-year follow-up. We used multivariable logistic regression and linear mixed models adjusted for age, sex, ethnicity, education, tobacco use, scanner technology and study site.The odds of ILAs increased 1.77-fold per 40 ppb increment in NOx (95% CI 1.06 to 2.95, p = 0.03). There was an overall trend towards an association between higher exposure to NOx and greater progression of HAAs (0.45% annual increase in HAAs per 40 ppb increment in NOx; 95% CI -0.02 to 0.92, p = 0.06). Associations of ambient fine particulate matter (PM2.5), NOx and NO2 concentrations with progression of HAAs varied by race/ethnicity (p = 0.002, 0.007, 0.04, respectively, for interaction) and were strongest among non-Hispanic white people.We conclude that ambient air pollution exposures were associated with subclinical ILD.

High attenuation areas on chest computed tomography in community-dwelling adults: the MESA study.

Evidence suggests that lung injury, inflammation and extracellular matrix remodelling precede lung fibrosis in interstitial lung disease (ILD). We examined whether a quantitative measure of increased lung attenuation on computed tomography (CT) detects lung injury, inflammation and extracellular matrix remodelling in community-dwelling adults sampled without regard to respiratory symptoms or smoking.We measured high attenuation areas (HAA; percentage of lung voxels between -600 and -250 Hounsfield Units) on cardiac CT scans of adults enrolled in the Multi-Ethnic Study of Atherosclerosis.HAA was associated with higher serum matrix metalloproteinase-7 (mean adjusted difference 6.3% per HAA doubling, 95% CI 1.3-11.5), higher interleukin-6 (mean adjusted difference 8.8%, 95% CI 4.8-13.0), lower forced vital capacity (FVC) (mean adjusted difference -82 mL, 95% CI -119--44), lower 6-min walk distance (mean adjusted difference -40 m, 95% CI -1--80), higher odds of interstitial lung abnormalities at 9.5 years (adjusted OR 1.95, 95% CI 1.43-2.65), and higher all cause-mortality rate over 12.2 years (HR 1.58, 95% CI 1.39-1.79).High attenuation areas are associated with biomarkers of inflammation and extracellular matrix remodelling, reduced lung function, interstitial lung abnormalities, and a higher risk of death among community-dwelling adults.

Factors influencing time-location patterns and their impact on estimates of exposure: the Multi-Ethnic Study of Atherosclerosis and Air Pollution (MESA Air).

We assessed time-location patterns and the role of individual- and residential-level characteristics on these patterns within the Multi-Ethnic Study of Atherosclerosis and Air Pollution (MESA Air) cohort and also investigated the impact of individual-level time-location patterns on individual-level estimates of exposure to outdoor air pollution. Reported time-location patterns varied significantly by demographic factors such as age, gender, race/ethnicity, income, education, and employment status. On average, Chinese participants reported spending significantly more time indoors and less time outdoors and in transit than White, Black, or Hispanic participants. Using a tiered linear regression approach, we predicted time indoors at home and total time indoors. Our model, developed using forward-selection procedures, explained 43% of the variability in time spent indoors at home, and incorporated demographic, health, lifestyle, and built environment factors. Time-weighted air pollution predictions calculated using recommended time indoors from USEPA overestimated exposures as compared with predictions made with MESA Air participant-specific information. These data fill an important gap in the literature by describing the impact of individual and residential characteristics on time-location patterns and by demonstrating the impact of population-specific data on exposure estimates.

Sociodemographic Correlates of Cognition in the Multi-Ethnic Study of Atherosclerosis (MESA).

OBJECTIVE: To describe the methodology utilized to evaluate cognitive function in the Multi-Ethnic Study of Atherosclerosis (MESA) and to present preliminary results by age, sex, and race/ethnicity. DESIGN: Cross-sectional measurements of a prospective observational cohort. SETTING: Residents of 6 U.S. communities free of cardiovascular disease at baseline (2000-02). PARTICIPANTS: 4,591 adults who completed the fifth MESA clinical examination in 2011-12; mean age 70.3 (SD: 9.5) years, 53.1% women, 40.7% non-Hispanic white, 26.4% non-Hispanic black, 21.4% Hispanic, and 11.5% Chinese. MEASUREMENTS: The cognitive battery consisted of the Cognitive Abilities Screening Instrument (version 2) to evaluate global cognition, the Digit Symbol Code for processing speed and Digit Spans Forward and Backward to assess memory. Demographic, socioeconomic, and cultural covariates were also collected for descriptive statistics and multivariate modeling. RESULTS: Associations between socioeconomic factors and cognition revealed that age, race/ethnicity, education, occupational status, household income, health insurance type, household size, place of birth, years and generation in U.S., and the presence of the ApoE4 allele were significantly associated with performance on the cognitive tests, although patterns varied by specific test, racial/ethnicity, and sociocultural factors. CONCLUSION: As many of the influencing cultural and socioeconomic factors measured here are complex, multifactorial, and may not be adequately quantified, caution has been recommended with regard to comparison and interpretation of racial/ethnic group performance differences from these cross-sectional models. These data provide a baseline for future exams and more comprehensive longitudinal analyses of the contributions of subclinical and clinical diseases to cognitive function and decline.

Home and work neighbourhood environments in relation to body mass index: the Multi-Ethnic Study of Atherosclerosis (MESA).

BACKGROUND: Little is known about the neighbourhood characteristics of workplaces, the extent to which they are independently and synergistically correlated with residential environments, and their impact on health. METHODS: This study investigated cross-sectional relationships between home and workplace neighbourhood environments with body mass index (BMI) in 1503 working participants of the Multi-Ethnic Study of Atherosclerosis with mean age 59.6 (SD=7.4). Neighbourhood features were socioeconomic status (SES), social environment (aesthetic quality, safety and social cohesion) and physical environment (walking environment, recreational facilities and food stores) derived from census data, locational data on businesses and survey data. Paired t tests and correlations compared environments overall and by distance between locations. Cross-classified multilevel models estimated associations with BMI. RESULTS: Home neighbourhoods had more favourable social environments while workplaces had more favourable SES and physical environments. Workplace and home measures were correlated (0.39-0.70), and differences between home and workplaces were larger as distance increased. Associations between BMI and neighbourhood SES and recreational facilities were stronger for home environment (p≤0.05) but did not significantly differ for healthy food, safety or social cohesion. Healthy food availability at home and work appeared to act synergistically (interaction p=0.01). CONCLUSIONS: Consideration of workplace environment may enhance our understanding of how place affects BMI.

Population structure of Hispanics in the United States: the multi-ethnic study of atherosclerosis.

Using ~60,000 SNPs selected for minimal linkage disequilibrium, we perform population structure analysis of 1,374 unrelated Hispanic individuals from the Multi-Ethnic Study of Atherosclerosis (MESA), with self-identification corresponding to Central America (n = 93), Cuba (n = 50), the Dominican Republic (n = 203), Mexico (n = 708), Puerto Rico (n = 192), and South America (n = 111). By projection of principal components (PCs) of ancestry to samples from the HapMap phase III and the Human Genome Diversity Panel (HGDP), we show the first two PCs quantify the Caucasian, African, and Native American origins, while the third and fourth PCs bring out an axis that aligns with known South-to-North geographic location of HGDP Native American samples and further separates MESA Mexican versus Central/South American samples along the same axis. Using k-means clustering computed from the first four PCs, we define four subgroups of the MESA Hispanic cohort that show close agreement with self-identification, labeling the clusters as primarily Dominican/Cuban, Mexican, Central/South American, and Puerto Rican. To demonstrate our recommendations for genetic analysis in the MESA Hispanic cohort, we present pooled and stratified association analysis of triglycerides for selected SNPs in the LPL and TRIB1 gene regions, previously reported in GWAS of triglycerides in Caucasians but as yet unconfirmed in Hispanic populations. We report statistically significant evidence for genetic association in both genes, and we further demonstrate the importance of considering population substructure and genetic heterogeneity in genetic association studies performed in the United States Hispanic population.

Analysis of family- and population-based samples in cohort genome-wide association studies.

Cohort studies typically sample unrelated individuals from a population, although family members of index cases may also be recruited to investigate shared familial risk factors. Recruitment of family members may be incomplete or ancillary to the main cohort, resulting in a mixed sample of independent family units, including unrelated singletons and multiplex families. Multiple methods are available to perform genome-wide association (GWA) analysis of binary or continuous traits in families, but it is unclear whether methods known to perform well on ascertained pedigrees, sibships, or trios are appropriate in analysis of a mixed unrelated cohort and family sample. We present simulation studies based on Multi-Ethnic Study of Atherosclerosis (MESA) pedigree structures to compare the performance of several popular methods of GWA analysis for both quantitative and dichotomous traits in cohort studies. We evaluate approaches suitable for analysis of families, and combined the best performing methods with population-based samples either by meta-analysis, or by pooled analysis of family- and population-based samples (mega-analysis), comparing type 1 error and power. We further assess practical considerations, such as availability of software and ability to incorporate covariates in statistical modeling, and demonstrate our recommended approaches through quantitative and binary trait analysis of HDL cholesterol (HDL-C) in 2,553 MESA family- and population-based African-American samples. Our results suggest linear modeling approaches that accommodate family-induced phenotypic correlation (e.g., variance-component model for quantitative traits or generalized estimating equations for dichotomous traits) perform best in the context of combined family- and population-based cohort GWAS.

Phenotype harmonization and cross-study collaboration in GWAS consortia: the GENEVA experience.

Genome-wide association study (GWAS) consortia and collaborations formed to detect genetic loci for common phenotypes or investigate gene-environment (G*E) interactions are increasingly common. While these consortia effectively increase sample size, phenotype heterogeneity across studies represents a major obstacle that limits successful identification of these associations. Investigators are faced with the challenge of how to harmonize previously collected phenotype data obtained using different data collection instruments which cover topics in varying degrees of detail and over diverse time frames. This process has not been described in detail. We describe here some of the strategies and pitfalls associated with combining phenotype data from varying studies. Using the Gene Environment Association Studies (GENEVA) multi-site GWAS consortium as an example, this paper provides an illustration to guide GWAS consortia through the process of phenotype harmonization and describes key issues that arise when sharing data across disparate studies. GENEVA is unusual in the diversity of disease endpoints and so the issues it faces as its participating studies share data will be informative for many collaborations. Phenotype harmonization requires identifying common phenotypes, determining the feasibility of cross-study analysis for each, preparing common definitions, and applying appropriate algorithms. Other issues to be considered include genotyping timeframes, coordination of parallel efforts by other collaborative groups, analytic approaches, and imputation of genotype data. GENEVA's harmonization efforts and policy of promoting data sharing and collaboration, not only within GENEVA but also with outside collaborations, can provide important guidance to ongoing and new consortia.

The Gene, Environment Association Studies consortium (GENEVA): maximizing the knowledge obtained from GWAS by collaboration across studies of multiple conditions.

Genome-wide association studies (GWAS) have emerged as powerful means for identifying genetic loci related to complex diseases. However, the role of environment and its potential to interact with key loci has not been adequately addressed in most GWAS. Networks of collaborative studies involving different study populations and multiple phenotypes provide a powerful approach for addressing the challenges in analysis and interpretation shared across studies. The Gene, Environment Association Studies (GENEVA) consortium was initiated to: identify genetic variants related to complex diseases; identify variations in gene-trait associations related to environmental exposures; and ensure rapid sharing of data through the database of Genotypes and Phenotypes. GENEVA consists of several academic institutions, including a coordinating center, two genotyping centers and 14 independently designed studies of various phenotypes, as well as several Institutes and Centers of the National Institutes of Health led by the National Human Genome Research Institute. Minimum detectable effect sizes include relative risks ranging from 1.24 to 1.57 and proportions of variance explained ranging from 0.0097 to 0.02. Given the large number of research participants (N>80,000), an important feature of GENEVA is harmonization of common variables, which allow analyses of additional traits. Environmental exposure information available from most studies also enables testing of gene-environment interactions. Facilitated by its sizeable infrastructure for promoting collaboration, GENEVA has established a unified framework for genotyping, data quality control, analysis and interpretation. By maximizing knowledge obtained through collaborative GWAS incorporating environmental exposure information, GENEVA aims to enhance our understanding of disease etiology, potentially identifying opportunities for intervention.

Paradoxical homozygous expression from heterozygotes and heterozygous expression from homozygotes as a consequence of transcriptional infidelity through a polyadenine tract in the AP3B1 gene responsible for canine cyclic neutropenia.

Canine cyclic neutropenia is an autosomal recessive disease in which the number of neutrophils, the primary blood phagocyte, oscillates between almost zero and normal values with two week frequency. We previously found that the causative mutation is an insertion of an extra adenine residue within a tract of nine A's in exon 21 of the 27 exon canine AP3B1 gene. In the course of identifying the mutation, however, we observed an unusual phenomenon: heterozygous carrier dogs, who have one normal allele and one mutant allele, produce a homogeneous population of normal AP3B1 transcripts (containing nine A's), but homozygous affected dogs, who have two mutant alleles, produce a heterogeneous population of AP3B1 mRNA containing mutant transcripts with ten A's and, unexpectedly, wild-type transcripts with nine A's. By RT-PCR subclone analysis and use of an in vitro reporter assay, we show that there is a high frequency of errors made during the transcription of homopolymeric adenine sequences, such that the A tract in the mRNA is frequently shortened or lengthened by an extra residue. Out of frame transcripts are degraded, accounting for this paradox through the preferential accumulation of normal message from mutant alleles.

A novel notch protein, N2N, targeted by neutrophil elastase and implicated in hereditary neutropenia.

Mutations in ELA2, encoding the human serine protease neutrophil elastase, cause cyclic and severe congenital neutropenia, and recent evidence indicates that the mutations alter the membrane trafficking of neutrophil elastase. These disorders feature impaired bone marrow production of neutrophils along with excess monocytes-terminally differentiated lineages corresponding to the two alternative fates of myeloid progenitor cells. We utilized a modified yeast two-hybrid system and identified a new, widely expressed gene, N2N, whose product is homologous to Notch2, that interacts with neutrophil elastase. N2N is a 36-kDa protein distributed throughout the cell and secreted. Its amino-terminal sequence consists of several EGF repeats identical to those of the extracellular region of Notch2, and its carboxyl terminus contains a unique 24-residue domain required for interaction with neutrophil elastase. Neutrophil elastase cleaves N2N within EGF repeats in vitro and in living cells, but the C-terminal domain retards proteolysis. In vitro, N2N represses transcriptional activities of Notch proteins. Disease-causing mutations of neutrophil elastase disrupt the interaction with N2N, impair proteolysis of N2N and Notch2, and interfere with Notch2 signaling, suggesting defective proteolysis of an inhibitory form of Notch as an explanation for the alternate switching of cell fates characteristic of hereditary neutropenia.

Lymphoid enhancer factor-1 links two hereditary leukemia syndromes through core-binding factor alpha regulation of ELA2.

Two hereditary human leukemia syndromes are severe congenital neutropenia (SCN), caused by mutations in the gene ELA2, encoding the protease neutrophil elastase, and familial platelet disorder with acute myelogenous leukemia (AML), caused by mutations in the gene AML1, encoding the transcription factor core-binding factor alpha (CBFalpha). In mice, CBFalpha regulates the expression of ELA2, suggesting a common link for both diseases. However, gene-targeted mouse models have failed to reproduce either human disease, thus prohibiting further in vivo studies in mice. Here we investigate CBFalpha regulation of the human ELA2 promoter, taking advantage of bone marrow obtained from patients with either illness. In particular, we have identified novel ELA2 promoter substitutions (-199 C to A) within a potential motif for lymphoid enhancer factor-1 (LEF-1), a transcriptional mediator of Wnt/beta-catenin signaling, in SCN patients. The LEF-1 motif lies adjacent to a potential CBFalpha binding site that is in a different position in human compared with mouse ELA2. We find that LEF-1 and CBFalpha co-activate ELA2 expression. In vitro, the high mobility group domain of LEF-1 interacts with the runt DNA binding and proline-, serine-, threonine-rich activation domains of CBFalpha. ELA2 transcript levels are up-regulated in bone marrow of an SCN patient with the -199 C to A substitution. Conversely, a mutation of the CBFalpha activation domain, found in a patient with familial platelet disorder with AML, fails to stimulate the ELA2 promoter in vitro, and bone marrow correspondingly demonstrates reduced ELA2 transcript. Observations in these complementary patients indicate that LEF-1 cooperates with CBFalpha to activate ELA2 in vivo and also suggest the possibility that up-regulating promoter mutations can contribute to SCN. Two hereditary AML predisposition syndromes may therefore intersect via LEF-1, potentially linking them to more generalized cancer mechanisms.

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase.

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) beta-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

Role of neutrophil elastase in bone marrow failure syndromes: molecular genetic revival of the chalone hypothesis.

Two forms of inherited deficiency of neutrophil numbers are cyclic hematopoiesis and severe congenital neutropenia. In cyclic hematopoiesis, neutrophil counts oscillate opposite monocytes in a 3-week cycle. Severe congenital neutropenia consists of static neutropenia and a predisposition to myelodysplasia and acute myelogenous leukemia. All cases of cyclic neutropenia and most cases of severe congenital neutropenia result from heterozygous germline mutations in the gene encoding neutrophil elastase, ela2. Recent work extends the list of neutropenia genes to include WASp, Gfi-1, adaptin, and tafazzin. Studies of mosaic patients suggest that ela2 mutations act in a cell-autonomous fashion. A hypothetical feedback circuit potentially interconnects these genes. Genetic dissection of signaling in model organisms along with experimental hematology implicate C/EPBepsilon, RUNX1/AML1, Notch family members, LEF1, and Cdc42 as additional nodes in this pathway. The authors propose that neutrophil elastase acts as an inhibitor of myelopoiesis, substantiating a chalone hypothesis proposed many years ago.