Impaired Kidney Function, Cerebral Small Vessel Disease and Cognitive Disorders: The Framingham Heart Study.

BACKGROUND AND HYPOTHESIS: It remains unclear if the relation of chronic kidney disease (CKD) with cognitive dysfunction is independent of blood pressure (BP). We evaluated kidney function in relation to premorbid BP measurements, cerebral small vessel disease (CSVD) and incident mild cognitive impairment (MCI) and dementia in Framingham Offspring Cohort participants. METHODS: We included Framingham Offspring participants free of dementia, attending an examination during midlife (exam cycle 6, baseline) for ascertainment of kidney function status, with brain MRI late in life (exam cycles 7-9), cognitive outcome data and available interim hypertension and blood pressure assessments. We related CKD (estimated glomerular filtration rate < 60 ml/min/1.73m2) and albuminuria (urine albumin-to-creatinine ratio ≥ 30 mg/g) to CSVD markers and cognitive outcomes using multivariable regression analyses. RESULTS: Among 2604 participants (mean age 67.4 ± 9.2, 64% women, 7% had CKD and 9% albuminuria), albuminuria was independently associated with covert infarcts (adjusted OR, 1.55 [1.00-2.38]; P = 0.049) and incident MCI and dementia (adjusted HR, 1.68 [1.18-2.41]; P = 0.005 and 1.71, [1.11-2.64]; P = 0.015, respectively). CKD was not associated with CSVD markers but was associated with higher risk of incident dementia (HR, 1.53 [1.02-2.29]; P = 0.041), While albuminuria was predictive of the Alzheimer's disease subtype (Adjusted HR = 1.68, [1.03-2.74]; P = 0.04), CKD was predictive of vascular dementia (Adjusted HR, 2.78, [1.16-6.68]; P = 0.023). CONCLUSIONS: Kidney disease was associated with CSVD and cognitive disorders in asymptomatic community dwelling participants. The relation was independent of premorbid BP, suggesting that the link between kidney and brain disease may involve additional mechanisms beyond blood pressure related injury.

Association of Apolipoprotein E ɛ4 Allele with Enlarged Perivascular Spaces.

OBJECTIVE: Enlarged perivascular spaces have emerged as markers of cerebral small vessel disease and are linked to perivascular drainage dysfunction. The apolipoprotein E-ɛ4 (APOE-ɛ4) allele is the strongest genetic risk factor for cerebral amyloid angiopathy and Alzheimer's neuropathology, but the underlying mechanisms remain unclear. We studied the relationship between APOE-ɛ4 and the topography and burden of enlarged perivascular spaces to elucidate underlying mechanisms between APOE-ɛ4 and adverse clinical outcomes. METHODS: We included 3,564 Framingham Heart Study participants with available genotypes and magnetic resonance imaging. Enlarged perivascular spaces in the basal ganglia and centrum semiovale were rated using a validated scale. We related APOE-ɛ4 allele presence to high burden of enlarged perivascular spaces in each region and a mixed score reflecting high burden in both regions using multivariable logistic regression. Exploratory analyses incorporated presence of cerebral microbleeds and assessed effect modification by hypertension. RESULTS: Mean age was 60.7 years (SD = 14.6), 1,644 (46.1%) were men, 1,486 (41.8%) were hypertensive, and 836 (23.5%) participants were APOE-ɛ4 carriers. APOE-ɛ4 was associated with high burden of enlarged perivascular spaces in the centrum semiovale (odds ratio [OR] = 1.45, 95% confidence interval [CI] = 1.16, 1.81) and mixed regions (OR = 1.37, 95% CI = 1.11, 1.68). Associations were slightly stronger in hypertensive subjects. INTERPRETATION: The APOE-ɛ4 allele plays a modest role in the burden of enlarged perivascular spaces in the centrum semiovale. Further studies are needed to clarify the underlying small vessel disease type in community-dwelling individuals with predominant centrum semiovale enlarged perivascular spaces, which may be hypertensive angiopathy in our sample. ANN NEUROL 2022;92:23-31.

Low Blood Pressure, Comorbidities, and Ischemic Stroke Mortality in US Veterans.

BACKGROUND AND PURPOSE: Low blood pressure (BP) is associated with higher stroke mortality, although the factors underlying this association have not been fully explored. We investigated prestroke BP and long-term mortality after ischemic stroke in a national sample of US veterans. METHODS: Using a retrospective cohort study design of veterans hospitalized between 2002 and 2007 with a first ischemic stroke and with ≥1 outpatient BP measurements 1 to 18 months before admission, we defined 6 categories each of average prestroke systolic BP (SBP) and diastolic BP, and 7 categories of pulse pressure. Patients were followed-up to 12 years for primary outcomes of all-cause and cardiovascular mortality. We used Cox models to relate prestroke BP indices to mortality and stratified analyses by the presence of preexisting comorbidities (smoking, myocardial infarction, heart failure, atrial fibrillation/flutter, cancer, and dementia), race and ethnicity. RESULTS: Of 29 690 eligible veterans with stroke (mean±SD age 67±12 years, 98% men, 67% White), 2989 (10%) had average prestroke SBP<120 mm Hg. During a follow-up of 4.1±3.3 years, patients with SBP<120 mm Hg experienced 61% all-cause and 27% cardiovascular mortality. In multivariable analyses, patients with the lowest SBP, lowest diastolic BP, and highest pulse pressure had the highest mortality risk: SBP<120 versus 130 to 139 mm Hg (hazard ratio=1.26 [95% CI, 1.19-1.34]); diastolic BP <60 versus 70 to 79 mm Hg (hazard ratio=1.35 [95% CI, 1.23-1.49]); and pulse pressure ≥90 versus 60 to 69 mm Hg (hazard ratio=1.24 [95% CI, 1.15-1.35]). Patients with average SBP<120 mm Hg and at least one comorbidity (smoking, heart disease, cancer, or dementia) had the highest mortality risk (hazard ratio=1.45 [95% CI, 1.37-1.53]). CONCLUSIONS: Compared with normotension, low prestroke BP was associated with mortality after stroke, particularly among patients with at least one comorbidity.

Circulating Monocyte Chemoattractant Protein-1 and Risk of Stroke: Meta-Analysis of Population-Based Studies Involving 17 180 Individuals.

Rationale: Proinflammatory cytokines have been identified as potential targets for lowering vascular risk. Experimental evidence and Mendelian randomization suggest a role of MCP-1 (monocyte chemoattractant protein-1) in atherosclerosis and stroke. However, data from large-scale observational studies are lacking. Objective: To determine whether circulating levels of MCP-1 are associated with risk of incident stroke in the general population. Methods and Results: We used previously unpublished data on 17 180 stroke-free individuals (mean age, 56.7±8.1 years; 48.8% men) from 6 population-based prospective cohort studies and explored associations between baseline circulating MCP-1 levels and risk of any stroke, ischemic stroke, and hemorrhagic stroke during a mean follow-up interval of 16.3 years (280 522 person-years at risk; 1435 incident stroke events). We applied Cox proportional-hazards models and pooled hazard ratios (HRs) using random-effects meta-analyses. After adjustments for age, sex, race, and vascular risk factors, higher MCP-1 levels were associated with increased risk of any stroke (HR per 1-SD increment in ln-transformed MCP-1, 1.07; 95% CI, 1.01-1.14). Focusing on stroke subtypes, we found a significant association between baseline MCP-1 levels and higher risk of ischemic stroke (HR, 1.11 [1.02-1.21]) but not hemorrhagic stroke (HR, 1.02 [0.82-1.29]). The results followed a dose-response pattern with a higher risk of ischemic stroke among individuals in the upper quartiles of MCP-1 levels as compared with the first quartile (HRs, second quartile: 1.19 [1.00-1.42]; third quartile: 1.35 [1.14-1.59]; fourth quartile: 1.38 [1.07-1.77]). There was no indication for heterogeneity across studies, and in a subsample of 4 studies (12 516 individuals), the risk estimates were stable after additional adjustments for circulating levels of IL (interleukin)-6 and high-sensitivity CRP (C-reactive protein). Conclusions: Higher circulating levels of MCP-1 are associated with increased long-term risk of stroke. Our findings along with genetic and experimental evidence suggest that MCP-1 signaling might represent a therapeutic target to lower stroke risk.Visual Overview: An online visual overview is available for this article.

Clustering of temporal gene expression data with mixtures of mixed effects models with a penalized likelihood.

MOTIVATION: Clustering algorithms like K-Means and standard Gaussian mixture models (GMM) fail to account for the structure of variability of replicated data or repeated measures over time. Additionally, a priori cluster number assumptions add an additional complexity to the process. Current methods to optimize cluster labels and number can be inaccurate or computationally intensive for temporal gene expression data with this additional variability. RESULTS: An extension to a model-based clustering algorithm is proposed using mixtures of mixed effects polynomial regression models and the EM algorithm with an entropy penalized log-likelihood function (EPEM). The EPEM is used to cluster temporal gene expression data with this additional variability. The addition of random effects in our model decreased the misclassification error when compared to mixtures of fixed effects models or other methods such as K-Means and GMM. Applying our method to microarray data from a fracture healing study revealed distinct temporal patterns of gene expression. AVAILABILITY AND IMPLEMENTATION: https://github.com/darlenelu72/EPEM-GMM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Discovery of Genetic Variation on Chromosome 5q22 Associated with Mortality in Heart Failure.

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7x10-9). We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5x10-40) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1x10-4). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure.

Targeted sequencing of genome wide significant loci associated with bone mineral density (BMD) reveals significant novel and rare variants: the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) targeted sequencing study.

Background: Bone mineral density (BMD) is a heritable phenotype that predicts fracture risk. We performed fine-mapping by targeted sequencing at WLS, MEF2C, ARHGAP1/F2 and JAG1 loci prioritized by eQTL and bioinformatic approaches among 56 BMD loci from our previous GWAS meta-analysis. Targeted sequencing was conducted in 1,291 Caucasians from the Framingham Heart Study ( n = 925) and Cardiovascular Health Study ( n = 366), including 206 women and men with extreme low femoral neck (FN) BMD. A total of 4,964 sequence variants (SNVs) were observed and 80% were rare with MAF <1%. The associations between previously identified SNPs in these loci and BMD, while nominally significant in sequenced participants, were no longer significant after multiple testing corrections. Conditional analyses did not find protein-coding variants that may be responsible for GWAS signals. On the other hand, in the sequenced subjects, we identified novel associations in WLS , ARHGAP1 , and 5' of MEF2C ( P- values < 8x10 - 5 ; false discovery rate (FDR) q-values < 0.01) that were much more strongly associated with BMD compared to the GWAS SNPs. These associated SNVs are less-common; independent from previous GWAS signals in the same loci; and located in gene regulatory elements. Our findings suggest that protein-coding variants in selected GWAS loci did not contribute to GWAS signals. By performing targeted sequencing in GWAS loci, we identified less-common and rare non-coding SNVs associated with BMD independently from GWAS common SNPs, suggesting both common and less-common variants may associate with disease risks and phenotypes in the same loci.

Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks.

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121*], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited.

Meta-analysis of 49†549 individuals imputed with the 1000 Genomes Project reveals an exonic damaging variant in ANGPTL4 determining fasting TG levels.

BACKGROUND: So far, more than 170 loci have been associated with circulating lipid levels through genome-wide association studies (GWAS). These associations are largely driven by common variants, their function is often not known, and many are likely to be markers for the causal variants. In this study we aimed to identify more new rare and low-frequency functional variants associated with circulating lipid levels. METHODS: We used the 1000 Genomes Project as a reference panel for the imputations of GWAS data from ∼60 000 individuals in the discovery stage and ∼90 000 samples in the replication stage. RESULTS: Our study resulted in the identification of five new associations with circulating lipid levels at four loci. All four loci are within genes that can be linked biologically to lipid metabolism. One of the variants, rs116843064, is a damaging missense variant within the ANGPTL4 gene. CONCLUSIONS: This study illustrates that GWAS with high-scale imputation may still help us unravel the biological mechanism behind circulating lipid levels.

Mining the LIPG allelic spectrum reveals the contribution of rare and common regulatory variants to HDL cholesterol.

Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5' UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5' UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci.

Hypertension Trends and White Matter Brain Injury in the Offspring Framingham Heart Study Cohort.

BACKGROUND: Hypertension is the most potent stroke risk factor and is also related to cerebral small vessel disease. We studied the relation between mid-to-late-life hypertension trends and cerebral white matter injury in community-dwelling individuals from the FHS (Framingham Heart Study). METHODS: FHS Offspring cohort participants with available mid-life and late-life blood pressure measurements and brain magnetic resonance imaging were included. Multiple regression analyses were used to relate hypertension trends (normotension-normotension [reference], normotension-hypertension, and hypertension-hypertension) to white matter injury metrics on diffusion tensor imaging (free water, fractional anisotropy, and peak skeletonized mean diffusivity) and Fluid Attenuated Inversion Recovery (white matter hyperintensity volume) by different blood pressure cutoffs (130/80, 140/90, and 150/90 mm Hg). RESULTS: We included 1018 participants (mean age 47.3±7.4 years at mid-life and 73.2±7.3 at late-life). At the 140/90 mm Hg cutoff, the hypertension-hypertension trend was associated with higher free water (ß, 0.16 [95% CI, 0.03-0.30]; P=0.021) and peak skeletonized mean diffusivity (ß, 0.15 [95% CI, 0.01-0.29]; P=0.033). At a 130/80 mm Hg cutoff, the hypertension-hypertension trend had significantly higher free water (ß, 0.16 [95% CI, 0.01-0.30]; P=0.035); and the normotension-hypertension (ß, 0.24 [95% CI, 0.03-0.44]; P=0.027) and hypertension-hypertension (ß, 0.22 [95% CI, 0.04-0.41]; P=0.022) trends had significantly increased white matter hyperintensity volume. Exploratory stratified analysis showed effect modifications by APOE ɛ4 allele and age. CONCLUSIONS: Mid-to-late-life hypertension exposure is significantly associated with microstructural and to a lesser extent, visible white matter injury; the effects are observed at both conventional and lower blood pressure cutoffs and are associated with longer duration of hypertension.

Association of JAG1 with bone mineral density and osteoporotic fractures: a genome-wide association study and follow-up replication studies.

Bone mineral density (BMD), a diagnostic parameter for osteoporosis and a clinical predictor of fracture, is a polygenic trait with high heritability. To identify genetic variants that influence BMD in different ethnic groups, we performed a genome-wide association study (GWAS) on 800 unrelated Southern Chinese women with extreme BMD and carried out follow-up replication studies in six independent study populations of European descent and Asian populations including 18,098 subjects. In the meta-analysis, rs2273061 of the Jagged1 (JAG1) gene was associated with high BMD (p = 5.27 x 10(-8) for lumbar spine [LS] and p = 4.15 x 10(-5) for femoral neck [FN], n = 18,898). This SNP was further found to be associated with the low risk of osteoporotic fracture (p = 0.009, OR = 0.7, 95% CI 0.57-0.93, n = 1881). Region-wide and haplotype analysis showed that the strongest association evidence was from the linkage disequilibrium block 5, which included rs2273061 of the JAG1 gene (p = 8.52 x 10(-9) for LS and 3.47 x 10(-5) at FN). To assess the function of identified variants, an electrophoretic mobility shift assay demonstrated the binding of c-Myc to the "G" but not "A" allele of rs2273061. A mRNA expression study in both human bone-derived cells and peripheral blood mononuclear cells confirmed association of the high BMD-related allele G of rs2273061 with higher JAG1 expression. Our results identify the JAG1 gene as a candidate for BMD regulation in different ethnic groups, and it is a potential key factor for fracture pathogenesis.

Genetics of coronary artery calcification among African Americans, a meta-analysis.

BACKGROUND: Coronary heart disease (CHD) is the major cause of death in the United States. Coronary artery calcification (CAC) scores are independent predictors of CHD. African Americans (AA) have higher rates of CHD but are less well-studied in genomic studies. We assembled the largest AA data resource currently available with measured CAC to identify associated genetic variants. METHODS: We analyzed log transformed CAC quantity (ln(CAC + 1)), for association with ~2.5 million single nucleotide polymorphisms (SNPs) and performed an inverse-variance weighted meta-analysis on results for 5,823 AA from 8 studies. Heritability was calculated using family studies. The most significant SNPs among AAs were evaluated in European Ancestry (EA) CAC data; conversely, the significance of published SNPs for CAC/CHD in EA was queried within our AA meta-analysis. RESULTS: Heritability of CAC was lower in AA (~30%) than previously reported for EA (~50%). No SNP reached genome wide significance (p < 5E-08). Of 67 SNPs with p < 1E-05 in AA there was no evidence of association in EA CAC data. Four SNPs in regions previously implicated in CAC/CHD (at 9p21 and PHACTR1) in EA reached nominal significance for CAC in AA, with concordant direction. Among AA, rs16905644 (p = 4.08E-05) had the strongest association in the 9p21 region. CONCLUSIONS: While we observed substantial heritability for CAC in AA, we failed to identify loci for CAC at genome-wide significant levels despite having adequate power to detect alleles with moderate to large effects. Although suggestive signals in AA were apparent at 9p21 and additional CAC and CAD EA loci, overall the data suggest that even larger samples and an ethnic specific focus will be required for GWAS discoveries for CAC in AA populations.

An integration of genome-wide association study and gene expression profiling to prioritize the discovery of novel susceptibility Loci for osteoporosis-related traits.

Osteoporosis is a complex disorder and commonly leads to fractures in elderly persons. Genome-wide association studies (GWAS) have become an unbiased approach to identify variations in the genome that potentially affect health. However, the genetic variants identified so far only explain a small proportion of the heritability for complex traits. Due to the modest genetic effect size and inadequate power, true association signals may not be revealed based on a stringent genome-wide significance threshold. Here, we take advantage of SNP and transcript arrays and integrate GWAS and expression signature profiling relevant to the skeletal system in cellular and animal models to prioritize the discovery of novel candidate genes for osteoporosis-related traits, including bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN), as well as geometric indices of the hip (femoral neck-shaft angle, NSA; femoral neck length, NL; and narrow-neck width, NW). A two-stage meta-analysis of GWAS from 7,633 Caucasian women and 3,657 men, revealed three novel loci associated with osteoporosis-related traits, including chromosome 1p13.2 (RAP1A, p = 3.6x10(-8)), 2q11.2 (TBC1D8), and 18q11.2 (OSBPL1A), and confirmed a previously reported region near TNFRSF11B/OPG gene. We also prioritized 16 suggestive genome-wide significant candidate genes based on their potential involvement in skeletal metabolism. Among them, 3 candidate genes were associated with BMD in women. Notably, 2 out of these 3 genes (GPR177, p = 2.6x10(-13); SOX6, p = 6.4x10(-10)) associated with BMD in women have been successfully replicated in a large-scale meta-analysis of BMD, but none of the non-prioritized candidates (associated with BMD) did. Our results support the concept of our prioritization strategy. In the absence of direct biological support for identified genes, we highlighted the efficiency of subsequent functional characterization using publicly available expression profiling relevant to the skeletal system in cellular or whole animal models to prioritize candidate genes for further functional validation.

Inflammatory biomarkers and MRI visible perivascular spaces: The Framingham Heart Study.

We studied the association between inflammatory biomarkers and magnetic resonance imaging (MRI) visible perivascular spaces (PVS) in Framingham Heart Study participants free of stroke and dementia. PVS in the basal ganglia (BG) and centrum semiovale (CSO) were rated with validated methods and categorized based on counts. A mixed score of high PVS burden in neither, one or both regions was also evaluated. We related biomarkers representing various inflammatory mechanisms to PVS burden using multivariable ordinal logistic regression analysis accounting for vascular risk factors and other MRI markers of cerebral small vessel disease. Among 3604 participants (mean age 58±13 years, 47% males), significant associations were observed for intercellular adhesion molecule1, fibrinogen, osteoprotegerin, and P-selectin in relation to BG PVS, P-selectin for CSO PVS, and tumor necrosis factor receptor 2, osteoprotegerin and cluster of differentiation 40 ligand for mixed topography PVS. Therefore, inflammation may have a role in the pathogenesis of cerebral small vessel disease and perivascular drainage dysfunction represented by PVS, with different and shared inflammatory biomarkers depending on PVS topography.

Oxidative metabolism is impaired by phosphate deficiency during fracture healing and is mechanistically related to BMP induced chondrocyte differentiation.

Prior studies of acute phosphate restriction during the endochondral phase of fracture healing showed delayed chondrocyte differentiation was mechanistically linked to decreased bone morphogenetic protein signaling. In the present study, transcriptomic analysis of fracture callus gene expression in three strains of mice was used to identify differentially expressed (FDR = q ≤ 0.05) genes in response to phosphate (Pi) restriction. Ontology and pathway analysis of these genes showed that independent of genetic background, a Pi-deficient diet downregulated (p = 3.16 × 10-23) genes associated with mitochondrial oxidative phosphorylation pathways as well as multiple other pathways of intermediate metabolism. Temporal clustering was used to identify co-regulation of these specific pathways. This analysis showed that specific Ox/Phos, tricarboxylic acid cycle, pyruvate dehydrogenase. Arginine, proline metabolism genes, and prolyl 4-hydroxylase were all co-regulated in response to dietary Pi restriction. The murine C3H10T½ mesenchymal stem cell line was used to assess the functional relationships between BMP2-induced chondrogenic differentiation, oxidative metabolism and extracellular matrix formation. BMP2-induced chondrogenic differentiation of C3H10T½ was carried out in culture media in the absence or presence of ascorbic acid, the necessary co-factor for proly hydroxylation, and in media with normal and 25 % phosphate levels. BMP2 treatment led to decreased proliferation, increased protein accumulation and increased collagen and aggrecan gene expression. Across all conditions, BMP2 increased total oxidative activity and ATP synthesis. Under all conditions, the presence of ascorbate further increased total protein accumulation, proly-hydroxylation and aggrecan gene expression, oxidative capacity and ATP production. Lower phosphate levels only diminished aggrecan gene expression with no other effects of metabolic activity being observed. These data suggest that dietary phosphate restriction controls endochondral growth in vivo indirectly through BMP signaling, which upregulates oxidative activity that is linked to overall protein production and collagen hydroxylation.

MRI Visible Perivascular Spaces and the Risk of Incident Mild Cognitive Impairment in a Community Sample.

BACKGROUND: Magnetic resonance imaging (MRI) visible perivascular spaces (PVS) are associated with the risk of incident dementia but their association with the early stages of cognitive impairment remains equivocal. OBJECTIVE: We examined the association between MRI visible PVS and the risk of incident mild cognitive impairment (MCI) in the community-based Framingham Heart Study (FHS). METHODS: FHS participants aged at least 50 years free of stroke, cognitive impairment, and dementia at the time of MRI were included. PVS were rated according to severity in the basal ganglia and centrum semiovale (CSO) using established criteria. Cox regression analyses were used to relate PVS to incident MCI adjusted for demographic and cardiovascular variables. RESULTS: The mean age of the sample (1,314 participants) at MRI was 68 years (SD, 9; 54% women). There were 263 cases of incident MCI over a median 7.4 years follow-up (max, 19.8 years). MCI risk increased with higher PVS severity in the CSO. Relative to persons with the lowest severity rating, persons with the highest severity rating in the CSO had a higher risk of incident MCI (hazard ratio [HR] = 2.55; 95% confidence interval [CI], 1.48-4.37; p = 0.0007). In secondary analysis, this association seemed stronger in women. Risk of incident MCI was nominally higher for participants with the highest severity grade of PVS in the basal ganglia, though not statistically significant relative to the lowest grade (HR = 2.19; 95% CI, 0.78-6.14; p = 0.14). CONCLUSIONS: PVS burden in the CSO may be a risk marker for early cognitive impairment.

Relation of MRI-Visible Perivascular Spaces and Other MRI Markers of Cerebral Small Vessel Disease.

Perivascular spaces (PVS) visible on brain MRI signal cerebral small vessel disease (CSVD). The coexistence of PVS with other CSVD manifestations likely increases the risk of adverse neurological outcomes. We related PVS to other CSVD manifestations and brain volumes that are markers of vascular brain injury and neurodegeneration. Framingham Heart Study (FHS) participants with CSVD ratings on brain MRI were included. PVS were rated in the basal ganglia (BG) and centrum semiovale (CSO) into grades I-IV and a category reflecting high burden in single or mixed CSO-BG regions. We related PVS to covert brain infarcts (CBI), white matter hyperintensities (WMH), cerebral microbleeds (CMB), total brain, hippocampal, and cortical gray matter volumes using adjusted multivariable regression analyses. In 2454 participants (mean age 54 ± 12 years), we observed that higher PVS burden in both BG and CSO was related to CMB in lobar and deep brain regions and increased WMH. Greater CSO PVS burden was associated with decreased total cortical gray volumes. PVS are associated with ischemic markers of CSVD and neurodegeneration markers. Further studies should elucidate the causality between PVS and other CSVD manifestations.

Association of MRI Visible Perivascular Spaces and Neurofilament Light Chain: The Framingham Heart Study.

BACKGROUND: Neurofilament light chain (NfL) is a marker of neuronal injury. Perivascular spaces (PVS) visible on magnetic resonance imaging (MRI) represent cerebral small vessel disease (CSVD) but their role as markers of neuronal injury needs further clarification. OBJECTIVE: To relate PVS burden according to brain topography and plasma NfL. METHODS: Framingham Heart Study (FHS) participants with brain MRI and NfL measurements were included. PVS were rated in the basal ganglia (BG) and centrum semiovale (CSO) using validated methods and categorized based on counts. A mixed region variable representing high burden PVS in either BG or CSO was assessed. Multivariable linear regression analyses were used to relate PVS burden to log-transformed NfL levels in models adjusted for age, sex, FHS cohort, time between MRI and clinic exam, and image view (model 1), vascular risk factors (model 2), and white matter hyperintensity volume, covert brain infarcts, and cerebral microbleeds (model 3). RESULTS: Among 1,457 participants (68.1±8.5 years, 45% males), NfL levels increased with higher PVS burden. Multivariable analysis showed an association of high PVS burden strictly in BG with NfL (ß= 0.117, 95% CI 0.014-0.221; p = 0.027), but attenuated in model 3. The associations were mainly in participants≥65 years (ß= 0.122, 95% CI 0.015-0.229, p = 0.026), women (ß= 0.156, 95% CI 0.024-0.288, p = 0.021), and APOE ɛ4 non-carriers (ß= 0.140, 95% CI 0.017-0.263, p = 0.026). CONCLUSIONS: The association of strictly BG high PVS burden with NfL suggests a role for PVS as markers of neuroaxonal injury, but our results are hypothesis generating and require further replication.

Bias due to two-stage residual-outcome regression analysis in genetic association studies.

Association studies of risk factors and complex diseases require careful assessment of potential confounding factors. Two-stage regression analysis, sometimes referred to as residual- or adjusted-outcome analysis, has been increasingly used in association studies of single nucleotide polymorphisms (SNPs) and quantitative traits. In this analysis, first, a residual-outcome is calculated from a regression of the outcome variable on covariates and then the relationship between the adjusted-outcome and the SNP is evaluated by a simple linear regression of the adjusted-outcome on the SNP. In this article, we examine the performance of this two-stage analysis as compared with multiple linear regression (MLR) analysis. Our findings show that when a SNP and a covariate are correlated, the two-stage approach results in biased genotypic effect and loss of power. Bias is always toward the null and increases with the squared-correlation between the SNP and the covariate (). For example, for , 0.1, and 0.5, two-stage analysis results in, respectively, 0, 10, and 50% attenuation in the SNP effect. As expected, MLR was always unbiased. Since individual SNPs often show little or no correlation with covariates, a two-stage analysis is expected to perform as well as MLR in many genetic studies; however, it produces considerably different results from MLR and may lead to incorrect conclusions when independent variables are highly correlated. While a useful alternative to MLR under , the two -stage approach has serious limitations. Its use as a simple substitute for MLR should be avoided.