Genome-wide association with diabetes-related traits in the Framingham Heart Study

Background

Susceptibility to type 2 diabetes may be conferred by genetic variants having modest effects on risk. Genome-wide fixed marker arrays offer a novel approach to detect these variants.

Methods

We used the Affymetrix 100K SNP array in 1,087 Framingham Offspring Study family members to examine genetic associations with three diabetes-related quantitative glucose traits (fasting plasma glucose (FPG), hemoglobin A1c, 28-yr time-averaged FPG (tFPG)), three insulin traits (fasting insulin, HOMA-insulin resistance, and 0–120 min insulin sensitivity index); and with risk for diabetes. We used additive generalized estimating equations (GEE) and family-based association test (FBAT) models to test associations of SNP genotypes with sex-age-age²-adjusted residual trait values, and Cox survival models to test incident diabetes.

Results

We found 415 SNPs associated (at p < 0.001) with at least one of the six quantitative traits in GEE, 242 in FBAT (18 overlapped with GEE for 639 non-overlapping SNPs), and 128 associated with incident diabetes (31 overlapped with the 639) giving 736 non-overlapping SNPs. Of these 736 SNPs, 439 were within 60 kb of a known gene. Additionally, 53 SNPs (of which 42 had r² < 0.80 with each other) had p < 0.01 for incident diabetes AND (all 3 glucose traits OR all 3 insulin traits, OR 2 glucose traits and 2 insulin traits); of these, 36 overlapped with the 736 other SNPs. Of 100K SNPs, one (rs7100927) was in moderate LD (r² = 0.50) with TCF7L2 (rs7903146), and was associated with risk of diabetes (Cox p-value 0.007, additive hazard ratio for diabetes = 1.56) and with tFPG (GEE p-value 0.03). There were no common (MAF > 1%) 100K SNPs in LD (r² > 0.05) with ABCC8 A1369S (rs757110), KCNJ11 E23K (rs5219), or SNPs in CAPN10 or HNFa. PPARG P12A (rs1801282) was not significantly associated with diabetes or related traits.

Conclusion

Framingham 100K SNP data is a resource for association tests of known and novel genes with diabetes and related traits posted at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. Framingham 100K data replicate the TCF7L2 association with diabetes.

     

Genome-wide association with select biomarker traits in the Framingham Heart Study

Background

Systemic biomarkers provide insights into disease pathogenesis, diagnosis, and risk stratification. Many systemic biomarker concentrations are heritable phenotypes. Genome-wide association studies (GWAS) provide mechanisms to investigate the genetic contributions to biomarker variability unconstrained by current knowledge of physiological relations.

Methods

We examined the association of Affymetrix 100K GeneChip single nucleotide polymorphisms (SNPs) to 22 systemic biomarker concentrations in 4 biological domains: inflammation/oxidative stress; natriuretic peptides; liver function; and vitamins. Related members of the Framingham Offspring cohort (n = 1012; mean age 59 ± 10 years, 51% women) had both phenotype and genotype data (minimum-maximum per phenotype n = 507–1008). We used Generalized Estimating Equations (GEE), Family Based Association Tests (FBAT) and variance components linkage to relate SNPs to multivariable-adjusted biomarker residuals. Autosomal SNPs (n = 70,987) meeting the following criteria were studied: minor allele frequency ≥ 10%, call rate ≥ 80% and Hardy-Weinberg equilibrium p ≥ 0.001.

Results

With GEE, 58 SNPs had p < 10^-6: the top SNPs were rs2494250 (p = 1.00*10^-14) and rs4128725 (p = 3.68*10^-12) for monocyte chemoattractant protein-1 (MCP1), and rs2794520 (p = 2.83*10^-8) and rs2808629 (p = 3.19*10^-8) for C-reactive protein (CRP) averaged from 3 examinations (over about 20 years). With FBAT, 11 SNPs had p < 10^-6: the top SNPs were the same for MCP1 (rs4128725, p = 3.28*10^-8, and rs2494250, p = 3.55*10^-8), and also included B-type natriuretic peptide (rs437021, p = 1.01*10^-6) and Vitamin K percent undercarboxylated osteocalcin (rs2052028, p = 1.07*10^-6). The peak LOD (logarithm of the odds) scores were for MCP1 (4.38, chromosome 1) and CRP (3.28, chromosome 1; previously described) concentrations; of note the 1.5 support interval included the MCP1 and CRP SNPs reported above (GEE model). Previous candidate SNP associations with circulating CRP concentrations were replicated at p < 0.05; the SNPs rs2794520 and rs2808629 are in linkage disequilibrium with previously reported SNPs. GEE, FBAT and linkage results are posted at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.

Conclusion

The Framingham GWAS represents a resource to describe potentially novel genetic influences on systemic biomarker variability. The newly described associations will need to be replicated in other studies.

     

Genome-wide association with bone mass and geometry in the Framingham Heart Study

Background

Osteoporosis is defined as the loss of bone mineral density that leads to bone fragility with aging. Population-based case-control studies have identified polymorphisms in many candidate genes that have been associated with bone mass maintenance or osteoporotic fracture. To investigate single nucleotide polymorphisms (SNPs) that are associated with osteoporosis, we examined the genetic variation among Koreans by analyzing 81 genes according to their function in bone formation and resorption during bone remodeling.

Methods

We resequenced all the exons, splice junctions and promoter regions of candidate osteoporosis genes using 24 unrelated Korean individuals. Using the common SNPs from our study and the HapMap database, a statistical analysis of deviation in heterozygosity depicted.

Results

We identified 942 variants, including 888 SNPs, 43 insertion/deletion polymorphisms, and 11 microsatellite markers. Of the SNPs, 557 (63%) had been previously identified and 331 (37%) were newly discovered in the Korean population. When compared SNPs in the Korean population with those in HapMap database, 1% (or less) of SNPs in the Japanese and Chinese subpopulations and 20% of those in Caucasian and African subpopulations were significantly differentiated from the Hardy-Weinberg expectations. In addition, an analysis of the genetic diversity showed that there were no significant differences among Korean, Han Chinese and Japanese populations, but African and Caucasian populations were significantly differentiated in selected genes. Nevertheless, in the detailed analysis of genetic properties, the LD and Haplotype block patterns among the five sub-populations were substantially different from one another.

Conclusion

Through the resequencing of 81 osteoporosis candidate genes, 118 unknown SNPs with a minor allele frequency (MAF) > 0.05 were discovered in the Korean population. In addition, using the common SNPs between our study and HapMap, an analysis of genetic diversity and deviation in heterozygosity was performed and the polymorphisms of the above genes among the five populations were substantially differentiated from one another. Further studies of osteoporosis could utilize the polymorphisms identified in our data since they may have important implications for the selection of highly informative SNPs for future association studies.     

Genome-wide association with bone mass and geometry in the Framingham Heart Study

Background

Osteoporosis is characterized by low bone mass and compromised bone structure, heritable traits that contribute to fracture risk. There have been no genome-wide association and linkage studies for these traits using high-density genotyping platforms.

Methods

We used the Affymetrix 100K SNP GeneChip marker set in the Framingham Heart Study (FHS) to examine genetic associations with ten primary quantitative traits: bone mineral density (BMD), calcaneal ultrasound, and geometric indices of the hip. To test associations with multivariable-adjusted residual trait values, we used additive generalized estimating equation (GEE) and family-based association tests (FBAT) models within each sex as well as sexes combined. We evaluated 70,987 autosomal SNPs with genotypic call rates ≥80%, HWE p ≥ 0.001, and MAF ≥10% in up to 1141 phenotyped individuals (495 men and 646 women, mean age 62.5 yrs). Variance component linkage analysis was performed using 11,200 markers.

Results

Heritability estimates for all bone phenotypes were 30–66%. LOD scores ≥3.0 were found on chromosomes 15 (1.5 LOD confidence interval: 51,336,679–58,934,236 bp) and 22 (35,890,398–48,603,847 bp) for femoral shaft section modulus. The ten primary phenotypes had 12 associations with 100K SNPs in GEE models at p < 0.000001 and 2 associations in FBAT models at p < 0.000001. The 25 most significant p-values for GEE and FBAT were all less than 3.5 × 10^-6 and 2.5 × 10^-5, respectively. Of the 40 top SNPs with the greatest numbers of significantly associated BMD traits (including femoral neck, trochanter, and lumbar spine), one half to two-thirds were in or near genes that have not previously been studied for osteoporosis. Notably, pleiotropic associations between BMD and bone geometric traits were uncommon. Evidence for association (FBAT or GEE p < 0.05) was observed for several SNPs in candidate genes for osteoporosis, such as rs1801133 in MTHFR; rs1884052 and rs3778099 in ESR1; rs4988300 in LRP5; rs2189480 in VDR; rs2075555 in COLIA1; rs10519297 and rs2008691 in CYP19, as well as SNPs in PPARG (rs10510418 and rs2938392) and ANKH (rs2454873 and rs379016). All GEE, FBAT and linkage results are provided as an open-access results resource at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.

Conclusion

The FHS 100K SNP project offers an unbiased genome-wide strategy to identify new candidate loci and to replicate previously suggested candidate genes for osteoporosis.

     

Genome-wide association study for subclinical atherosclerosis in major arterial territories in the NHLBI's Framingham Heart Study

Introduction

Subclinical atherosclerosis (SCA) measures in multiple arterial beds are heritable phenotypes that are associated with increased incidence of cardiovascular disease. We conducted a genome-wide association study (GWAS) for SCA measurements in the community-based Framingham Heart Study.

Methods

Over 100,000 single nucleotide polymorphisms (SNPs) were genotyped (Human 100K GeneChip, Affymetrix) in 1345 subjects from 310 families. We calculated sex-specific age-adjusted and multivariable-adjusted residuals in subjects tested for quantitative SCA phenotypes, including ankle-brachial index, coronary artery calcification and abdominal aortic calcification using multi-detector computed tomography, and carotid intimal medial thickness (IMT) using carotid ultrasonography. We evaluated associations of these phenotypes with 70,987 autosomal SNPs with minor allele frequency ≥ 0.10, call rate ≥ 80%, and Hardy-Weinberg p-value ≥ 0.001 in samples ranging from 673 to 984 subjects, using linear regression with generalized estimating equations (GEE) methodology and family-based association testing (FBAT). Variance components LOD scores were also calculated.

Results

There was no association result meeting criteria for genome-wide significance, but our methods identified 11 SNPs with p < 10^-5 by GEE and five SNPs with p < 10^-5 by FBAT for multivariable-adjusted phenotypes. Among the associated variants were SNPs in or near genes that may be considered candidates for further study, such as rs1376877 (GEE p < 0.000001, located in ABI2) for maximum internal carotid artery IMT and rs4814615 (FBAT p = 0.000003, located in PCSK2) for maximum common carotid artery IMT. Modest significant associations were noted with various SCA phenotypes for variants in previously reported atherosclerosis candidate genes, including NOS3 and ESR1. Associations were also noted of a region on chromosome 9p21 with CAC phenotypes that confirm associations with coronary heart disease and CAC in two recently reported genome-wide association studies. In linkage analyses, several regions of genome-wide linkage were noted, confirming previously reported linkage of internal carotid artery IMT on chromosome 12. All GEE, FBAT and linkage results are provided as an open-access results resource at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.

Conclusion

The results from this GWAS generate hypotheses regarding several SNPs that may be associated with SCA phenotypes in multiple arterial beds. Given the number of tests conducted, subsequent independent replication in a staged approach is essential to identify genetic variants that may be implicated in atherosclerosis.

     

Genome-wide association to body mass index and waist circumference: the Framingham Heart Study 100K project

Background

Obesity is related to multiple cardiovascular disease (CVD) risk factors as well as CVD and has a strong familial component. We tested for association between SNPs on the Affymetrix 100K SNP GeneChip and measures of adiposity in the Framingham Heart Study.

Methods

A total of 1341 Framingham Heart Study participants in 310 families genotyped with the Affymetrix 100K SNP GeneChip had adiposity traits measured over 30 years of follow up. Body mass index (BMI), waist circumference (WC), weight change, height, and radiographic measures of adiposity (subcutaneous adipose tissue, visceral adipose tissue, waist circumference, sagittal height) were measured at multiple examination cycles. Multivariable-adjusted residuals, adjusting for age, age-squared, sex, smoking, and menopausal status, were evaluated in association with the genotype data using additive Generalized Estimating Equations (GEE) and Family Based Association Test (FBAT) models. We prioritized mean BMI over offspring examinations (1–7) and cohort examinations (10, 16, 18, 20, 22, 24, 26) and mean WC over offspring examinations (4–7) for presentation. We evaluated associations with 70,987 SNPs on autosomes with minor allele frequencies of at least 0.10, Hardy-Weinberg equilibrium p ≥ 0.001, and call rates of at least 80%.

Results

The top SNPs to be associated with mean BMI and mean WC by GEE were rs110683 (p-value 1.22*10^-7) and rs4471028 (p-values 1.96*10^-7). Please see http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007 for the complete set of results. We were able to validate SNPs in known genes that have been related to BMI or other adiposity traits, including the ESR1 Xba1 SNP, PPARG, and ADIPOQ.

Conclusion

Adiposity traits are associated with SNPs on the Affymetrix 100K SNP GeneChip. Replication of these initial findings is necessary. These data will serve as a resource for replication as more genes become identified with BMI and WC.

     

Genome-wide association to body mass index and waist circumference: the Framingham Heart Study 100K project

Background

The FABP2 gene encodes for the intestinal FABP (IFABP) protein, which is expressed only in intestinal enterocytes. A polymorphism at codon 54 in exon 2 of the FABP2 gene exchanges an Alanine (Ala), in the small helical region of the protein, for Threonine (Thr). Given the potential physiological role of the Ala54Thr FABP2 polymorphism, we assess in this study the local population frequency and analyze possible associations with five selected markers, i.e. glycemia, total cholesterol, body mass index (BMI), hypertension, and high Cardiovascular Risk Index (CVR index).

Methods

We studied 86 men and 116 women. DNA was extracted from a blood drop for genotype analysis. Allele frequencies were calculated by direct counting. Hardy Weinberg Equilibrium was evaluated using a Chi-square goodness of fit test. For the polymorphism association analysis, five markers were selected, i.e. blood pressure, Framingham Risk Index, total cholesterol, BMI, and glycemia. For each marker, the Odds Ratio (OR) was calculated by an online statistic tool.

Results

Our results reveal a similar population polymorphism frequency as in previous European studies, with q = 0.277 (95% confidence limits 0.234–0.323). No significant association was found with any of the tested markers in the context of our Argentine nutritional and cultural habits. We did, however, observe a tendency for increased Cholesterol and high BMI in Thr54 carriers.

Conclusion

This is the first study to look at the population frequency of the Thr54 allele in Argentina. The obtained result does not differ from previously reported frequencies in European populations. Moreover, we found no association between the Thr54 allele and any of the five selected markers. The observed tendency to increased total cholesterol and elevated BMI in Thr54 carriers, even though not significant for p < 0.1 could be worth of further investigation to establish whether the Thr54 variant should be taken into consideration in cardiovascular prevention strategies.     

Genome-wide association and linkage analyses of hemostatic factors and hematological phenotypes in the Framingham Heart Study

Background

Increased circulating levels of hemostatic factors as well as anemia have been associated with increased risk of cardiovascular disease (CVD). Known associations between hemostatic factors and sequence variants at genes encoding these factors explain only a small proportion of total phenotypic variation. We sought to confirm known putative loci and identify novel loci that may influence either trait in genome-wide association and linkage analyses using the Affymetrix GeneChip 100K single nucleotide polymorphism (SNP) set.

Methods

Plasma levels of circulating hemostatic factors (fibrinogen, factor VII, plasminogen activator inhibitor-1, von Willebrand factor, tissue plasminogen activator, D-dimer) and hematological phenotypes (platelet aggregation, viscosity, hemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin concentration) were obtained in approximately 1000 Framingham Heart Study (FHS) participants from 310 families. Population-based association analyses using the generalized estimating equations (GEE), family-based association test (FBAT), and multipoint variance components linkage analyses were performed on the multivariable adjusted residuals of hemostatic and hematological phenotypes.

Results

In association analysis, the lowest GEE p-value for hemostatic factors was p = 4.5*10^-16 for factor VII at SNP rs561241, a variant located near the F7 gene and in complete linkage disequilibrium (LD) (r² = 1) with the Arg353Gln F7 SNP previously shown to account for 9% of total phenotypic variance. The lowest GEE p-value for hematological phenotypes was 7*10^-8 at SNP rs2412522 on chromosome 4 for mean corpuscular hemoglobin concentration. We presented top 25 most significant GEE results with p-values in the range of 10^-6 to 10^-5 for hemostatic or hematological phenotypes. In relating 100K SNPs to known candidate genes, we identified two SNPs (rs1582055, rs4897475) in erythrocyte membrane protein band 4.1-like 2 (EPB41L2) associated with hematological phenotypes (GEE p < 10^-3). In linkage analyses, the highest linkage LOD score for hemostatic factors was 3.3 for factor VII on chromosome 10 around 15 Mb, and for hematological phenotypes, LOD 3.4 for hemoglobin on chromosome 4 around 55 Mb. All GEE and FBAT association and variance components linkage results can be found at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007

Conclusion

Using genome-wide association methodology, we have successfully identified a SNP in complete LD with a sequence variant previously shown to be strongly associated with factor VII, providing proof of principle for this approach. Further study of additional strongly associated SNPs and linked regions may identify novel variants that influence the inter-individual variability in hemostatic factors and hematological phenotypes.

     

A genome-wide association for kidney function and endocrine-related traits in the NHLBI's Framingham Heart Study

Background

Glomerular filtration rate (GFR) and urinary albumin excretion (UAE) are markers of kidney function that are known to be heritable. Many endocrine conditions have strong familial components. We tested for association between the Affymetrix GeneChip Human Mapping 100K single nucleotide polymorphism (SNP) set and measures of kidney function and endocrine traits.

Methods

Genotype information on the Affymetrix GeneChip Human Mapping 100K SNP set was available on 1345 participants. Serum creatinine and cystatin-C (cysC; n = 981) were measured at the seventh examination cycle (1998–2001); GFR (n = 1010) was estimated via the Modification of Diet in Renal Disease (MDRD) equation; UAE was measured on spot urine samples during the sixth examination cycle (1995–1998) and was indexed to urinary creatinine (n = 822). Thyroid stimulating hormone (TSH) was measured at the third and fourth examination cycles (1981–1984; 1984–1987) and mean value of the measurements were used (n = 810). Age-sex-adjusted and multivariable-adjusted residuals for these measurements were used in association with genotype data using generalized estimating equations (GEE) and family-based association tests (FBAT) models. We presented the results for association tests using additive allele model. We evaluated associations with 70,987 SNPs on autosomes with minor allele frequencies of at least 0.10, Hardy-Weinberg Equilibrium p-value ≥ 0.001, and call rates of at least 80%.

Results

The top SNPs associated with these traits using the GEE method were rs2839235 with GFR (p-value 1.6*10^-05), rs1158167 with cysC (p-value 8.5*10^-09), rs1712790 with UAE (p-value 1.9*10^-06), and rs6977660 with TSH (p-value 3.7*10^-06), respectively. The top SNPs associated with these traits using the FBAT method were rs6434804 with GFR(p-value 2.4*10^-5), rs563754 with cysC (p-value 4.7*10^-5), rs1243400 with UAE (p-value 4.8*10^-6), and rs4128956 with TSH (p-value 3.6*10^-5), respectively. Detailed association test results can be found at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. Four SNPs in or near the CST3 gene were highly associated with cysC levels (p-value 8.5*10^-09 to 0.007).

Conclusion

Kidney function traits and TSH are associated with SNPs on the Affymetrix GeneChip Human Mapping 100K SNP set. These data will serve as a valuable resource for replication as more SNPs associated with kidney function and endocrine traits are identified.

     

Genome-wide association analysis of total cholesterol and high-density lipoprotein cholesterol levels using the Framingham Heart Study data

Background  

Cholesterol concentrations in blood are related to cardiovascular diseases. Recent genome-wide association studies (GWAS) of cholesterol levels identified a number of single-locus effects on total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels. Here, we report single-locus and epistasis SNP effects on TC and HDL-C using the Framingham Heart Study (FHS) data.     

Genome-wide association of echocardiographic dimensions,brachial artery endothelial function and treadmill exercise responses in the Framingham Heart Study

Background:

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; TNFRSF6), a cell surface receptor that regulates apoptosis and its signaling apparatus.

Methods:

Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death.

Results:

We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the protein's extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis.

Conclusion:

Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.     

Genome-wide association of echocardiographic dimensions,brachial artery endothelial function and treadmill exercise responses in the Framingham Heart Study

Background

Echocardiographic left ventricular (LV) measurements, exercise responses to standardized treadmill test (ETT) and brachial artery (BA) vascular function are heritable traits that are associated with cardiovascular disease risk. We conducted a genome-wide association study (GWAS) in the community-based Framingham Heart Study.

Methods

We estimated multivariable-adjusted residuals for quantitative echocardiography, ETT and BA function traits. Echocardiography residuals were averaged across 4 examinations and included LV mass, diastolic and systolic dimensions, wall thickness, fractional shortening, left atrial and aortic root size. ETT measures (single exam) included systolic blood pressure and heart rate responses during exercise stage 2, and at 3 minutes post-exercise. BA measures (single exam) included vessel diameter, flow-mediated dilation (FMD), and baseline and hyperemic flow responses. Generalized estimating equations (GEE), family-based association tests (FBAT) and variance-components linkage were used to relate multivariable-adjusted trait residuals to 70,987 SNPs (Human 100K GeneChip, Affymetrix) restricted to autosomal SNPs with minor allele frequency ≥0.10, genotype call rate ≥0.80, and Hardy-Weinberg equilibrium p ≥ 0.001.

Results

We summarize results from 17 traits in up to 1238 related middle-aged to elderly men and women. Results of all association and linkage analyses are web-posted at http://ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. We confirmed modest-to-strong heritabilities (estimates 0.30–0.52) for several Echo, ETT and BA function traits. Overall, p < 10^-5 in either GEE or FBAT models were observed for 21 SNPs (nine for echocardiography, eleven for ETT and one for BA function). The top SNPs associated were (GEE results): LV diastolic dimension, rs1379659 (SLIT2, p = 1.17*10^-7); LV systolic dimension, rs10504543 (KCNB2, p = 5.18*10^-6); LV mass, rs10498091 (p = 5.68*10^-6); Left atrial size, rs1935881 (FAM5C, p = 6.56*10^-6); exercise heart rate, rs6847149 (NOLA1, p = 2.74*10^-6); exercise systolic blood pressure, rs2553268 (WRN, p = 6.3*10^-6); BA baseline flow, rs3814219 (OBFC1, 9.48*10^-7), and FMD, rs4148686 (CFTR, p = 1.13*10^-5). Several SNPs are reasonable biological candidates, with some being related to multiple traits suggesting pleiotropy. The peak LOD score was for LV mass (4.38; chromosome 5); the 1.5 LOD support interval included NRG2.

Conclusion

In hypothesis-generating GWAS of echocardiography, ETT and BA vascular function in a moderate-sized community-based sample, we identified several SNPs that are candidates for replication attempts and we provide a web-based GWAS resource for the research community.

     

A genome-wide association study for blood lipid phenotypes in the Framingham Heart Study

Background

Blood lipid levels including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) are highly heritable. Genome-wide association is a promising approach to map genetic loci related to these heritable phenotypes.

Methods

In 1087 Framingham Heart Study Offspring cohort participants (mean age 47 years, 52% women), we conducted genome-wide analyses (Affymetrix 100K GeneChip) for fasting blood lipid traits. Total cholesterol, HDL-C, and TG were measured by standard enzymatic methods and LDL-C was calculated using the Friedewald formula. The long-term averages of up to seven measurements of LDL-C, HDL-C, and TG over a ~30 year span were the primary phenotypes. We used generalized estimating equations (GEE), family-based association tests (FBAT) and variance components linkage to investigate the relationships between SNPs (on autosomes, with minor allele frequency ≥10%, genotypic call rate ≥80%, and Hardy-Weinberg equilibrium p ≥ 0.001) and multivariable-adjusted residuals. We pursued a three-stage replication strategy of the GEE association results with 287 SNPs (P < 0.001 in Stage I) tested in Stage II (n ~1450 individuals) and 40 SNPs (P < 0.001 in joint analysis of Stages I and II) tested in Stage III (n~6650 individuals).

Results

Long-term averages of LDL-C, HDL-C, and TG were highly heritable (h² = 0.66, 0.69, 0.58, respectively; each P < 0.0001). Of 70,987 tests for each of the phenotypes, two SNPs had p < 10^-5 in GEE results for LDL-C, four for HDL-C, and one for TG. For each multivariable-adjusted phenotype, the number of SNPs with association p < 10^-4 ranged from 13 to 18 and with p < 10^-3, from 94 to 149. Some results confirmed previously reported associations with candidate genes including variation in the lipoprotein lipase gene (LPL) and HDL-C and TG (rs7007797; P = 0.0005 for HDL-C and 0.002 for TG). The full set of GEE, FBAT and linkage results are posted at the database of Genotype and Phenotype (dbGaP). After three stages of replication, there was no convincing statistical evidence for association (i.e., combined P < 10^-5 across all three stages) between any of the tested SNPs and lipid phenotypes.

Conclusion

Using a 100K genome-wide scan, we have generated a set of putative associations for common sequence variants and lipid phenotypes. Validation of selected hypotheses in additional samples did not identify any new loci underlying variability in blood lipids. Lack of replication may be due to inadequate statistical power to detect modest quantitative trait locus effects (i.e., <1% of trait variance explained) or reduced genomic coverage of the 100K array. GWAS in FHS using a denser genome-wide genotyping platform and a better-powered replication strategy may identify novel loci underlying blood lipids.

     

A genome-wide association study of breast and prostate cancer in the NHLBI's Framingham Heart Study

Background

Cutaneous Malignant Melanoma causes over 75% of skin cancer-related deaths, and it is clear that many factors may contribute to the outcome. Matrix Metalloproteinases (MMPs) play an important role in the degradation and remodeling of the extracellular matrix and basement membrane that, in turn, modulate cell division, migration and angiogenesis. Some polymorphisms are known to influence gene expression, protein activity, stability, and interactions, and they were shown to be associated with certain tumor phenotypes and cancer risk.

Methods

We tested seven polymorphisms within the MMP-9 gene in 1002 patients with melanoma in order to evaluate germline genetic variants and their association with progression and known risk factors of melanoma. The polymorphisms were selected based on previously published reports and their known or potential functional relevance using in-silico methods. Germline DNA was then genotyped using pyrosequencing, melting temperature profiles, heteroduplex analysis, and fragment size analysis.

Results

We found that reference alleles were present in higher frequency in patients who tend to sunburn, have family history of melanoma, higher melanoma stage, intransit metastasis and desmoplastic melanomas among others. However, after adjustment for age, sex, phenotypic index, moles, and freckles only Q279R, P574R and R668Q had significant associations with intransit metastasis, propensity to tan/sunburn and primary melanoma site.

Conclusion

This study does not provide strong evidence for further investigation into the role of the MMP-9 SNPs in melanoma progression.     

A genome-wide association study of breast and prostate cancer in the NHLBI's Framingham Heart Study

Background

Breast and prostate cancer are two commonly diagnosed cancers in the United States. Prior work suggests that cancer causing genes and cancer susceptibility genes can be identified.

Methods

We conducted a genome-wide association study (Affymetrix 100K SNP GeneChip) of cancer in the community-based Framingham Heart Study. We report on 2 cancer traits – prostate cancer and breast cancer – in up to 1335 participants from 330 families (54% women, mean entry age 33 years). Multivariable-adjusted residuals, computed using Cox proportional hazards models, were tested for association with qualifying SNPs (70, 987 autosomal SNPs with genotypic call rate ≥80%, minor allele frequency ≥10%, Hardy-Weinberg test p ≥ 0.001) using generalized estimating equations (GEE) models and family based association tests (FBAT).

Results

There were 58 women with breast cancer and 59 men with prostate cancer. No SNP associations attained genome-wide significance. The top SNP associations in GEE models for each trait were as follows: breast cancer, rs2075555, p = 8.0 × 10^-8 in COL1A1; and prostate cancer, rs9311171, p = 1.75 × 10^-6 in CTDSPL. In analysis of selected candidate cancer susceptibility genes, two MSR1 SNPs (rs9325782, GEE p = 0.008 and rs2410373, FBAT p = 0.021) were associated with prostate cancer and three ERBB4 SNPs (rs905883 GEE p = 0.0002, rs7564590 GEE p = 0.003, rs7558615 GEE p = 0.0078) were associated with breast cancer. The previously reported risk SNP for prostate cancer, rs1447295, was not included on the 100K chip. Results of cancer phenotype-genotype associations for all autosomal SNPs are web posted at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.

Conclusion

Although no association attained genome-wide significance, several interesting associations emerged for breast and prostate cancer. These findings can serve as a resource for replication in other populations to identify novel biologic pathways contributing to cancer susceptibility.

     

Framingham Heart Study genome-wide association: results for pulmonary function measures

Background

Pulmonary function measures obtained by spirometry are used to diagnose chronic obstructive pulmonary disease (COPD) and are highly heritable. We conducted genome-wide association (GWA) analyses (Affymetrix 100K SNP GeneChip) for measures of lung function in the Framingham Heart Study.

Methods

Ten spirometry phenotypes including percent of predicted measures, mean spirometry measures over two examinations, and rates of change based on forced expiratory volume in one second (FEV₁), forced vital capacity (FVC), forced expiratory flow from the 25^th to 75^th percentile (FEF_25–75), the FEV₁/FVC ratio, and the FEF_25–75/FVC ratio were examined. Percent predicted phenotypes were created using each participant's latest exam with spirometry. Predicted lung function was estimated using models defined in the set of healthy never-smokers, and standardized residuals of percent predicted measures were created adjusting for smoking status, pack-years, and body mass index (BMI). All modeling was performed stratified by sex and cohort. Mean spirometry phenotypes were created using data from two examinations and adjusting for age, BMI, height, smoking and pack-years. Change in pulmonary function over time was studied using two to four examinations with spirometry to calculate slopes, which were then adjusted for age, height, smoking and pack-years.

Results

Analyses were restricted to 70,987 autosomal SNPs with minor allele frequency ≥ 10%, genotype call rate ≥ 80%, and Hardy-Weinberg equilibrium p-value ≥ 0.001. A SNP in the interleukin 6 receptor (IL6R) on chromosome 1 was among the best results for percent predicted FEF_25–75. A non-synonymous coding SNP in glutathione S-transferase omega 2 (GSTO2) on chromosome 10 had top-ranked results studying the mean FEV₁ and FVC measurements from two examinations. SNPs nearby the SOD3 and vitamin D binding protein genes, candidate genes for COPD, exhibited association to percent predicted phenotypes.

Conclusion

GSTO2 and IL6R are credible candidate genes for association to pulmonary function identified by GWA. These and other observed associations warrant replication studies. This resource of GWA results for pulmonary function measures is publicly available at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.

     

Heart rate and heart rate variability in chicken embryos at the end of incubation

Our immediate goal was to study heart rate variability (HRV) in chicken embryos in the egg. Instantaneous heart rate data were needed for this purpose, and accordingly an ECG recording method in the egg was developed. The aim of this work was to test the hypothesis that autonomic nervous cardiac modulation, as shown from HRV parameters, is present at the end of development and that it reaches a constant value during the last days of incubation. Embryonic chicken heart rate was obtained at the final incubation period (days 19 and 20) from ECG recordings. Tachograms were computed and time- and frequency-domain indices of HRV were determined. No significant differences were found between HRV indices from day 19 and day 20. The power spectra extended in two frequency bands with centre frequency around 0.6-0.7 Hz (low frequency (LF) component), and another around 1.2-1.5 Hz (high frequency (HF) component); the latter was shown to reflect respiratory sinus arrhythmia. A relation between mean RR interval and some HRV parameters (rMSSD, pNN5 and HF power) was shown. HRV results obtained from embryonic chickens, showed the presence of modulation of cardiovascular function by the autonomic nervous system. The results suggested that sympathetic and parasympathetic activities have already reached a constant level at day 19 of incubation. High frequency oscillations (0.78-2.5 Hz) were detected and are considered to reflect respiratory sinus arrhythmia.