Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies.

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.

Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

Analysis of Variation in Clubfoot Candidate Genes

Isolated clubfoot, a common birth defect occurring in more than 135,000 livebirths worldwide each year, is associated with significant health care and financial burdens. Clubfoot is defined by forefoot adduction, hindfoot varus, midfoot cavus and hindfoot equinus. Isolated clubfoot, which is the focus of these studies, is distinct from syndromic clubfoot because there are no other associated malformations. Population, family, twin and segregation analysis studies provide evidence that genetic and environmental factors play an etiologic role in isolated clubfoot. The studies described in this thesis were performed to define the role of genetic variation in isolated clubfoot. Interrogation of a deletion region associated with syndromic clubfoot, suggested that CASP8 and CASP10, two apoptotic genes, play a role in isolated clubfoot. To explore the role of apoptotic genes in clubfoot, SNPs spanning genes involved in the apoptotic pathway in the six chromosomal deletion regions, and limb patterning genes, HOXD and HOXA, were interrogated. SNPs in mitochondrial mediated apoptotic genes and several SNPs in HOXA and HOXD genes were modestly associated with clubfoot with the most significant SNP, rs3801776, located in the basal promoter of HOXA9. Several significant associations were found with SNPs in NFAT2 and TNIP2. Significant gene interactions were detected between SNPs in HOX and apoptotic genes. These findings suggest a model for clubfoot in which variation in one gene is not sufficient to cause the malformation but requires variation several genes to perturb protein expression sufficiently to alter muscle and foot development. These results significantly impact our knowledge base by delineating underlying mechanisms causing clubfoot.

Xenobiotic Metabolism Genes and Clubfoot

Idiopathic or isolated clubfoot is a common orthopedic birth defect that affects approximately 135,000 children worldwide. It is characterized by equinus, varus and adductus deformities of the ankle and foot. Correction of clubfoot involves months of serial manipulations, castings and bracing, with surgical correction needed in forty percent of cases. Multifactorial etiology has been suggested in numerous studies with both environmental and genetic factors playing an etiologic role. Maternal smoking during pregnancy is the only common environmental factor that has consistently been shown to increase the risk for clubfoot. Moreover, a positive family history of clubfoot and maternal smoking increases the risk of clubfoot twenty fold. These findings suggest that genetic variation in smoking metabolism genes may increase susceptibility to clubfoot. Based on this reasoning, we interrogated eight candidate genes, chosen based on their involvement in phase 1 and 2 cigarette smoke metabolism. Twenty-two SNPs and two null alleles in eight genes (CYP1A1, CYP1A2, CYP1B1, CYP2A6, EPHX1, NAT2, GSTM1 and GSTT1) were genotyped in a dataset composed of nonHispanic white and Hispanic multiplex and simplex families. Only one SNP in CYP1A1, rs1048943, had significantly altered transmission in the aggregate and multiplex NHW datasets (p=0.003 and p=0.009). Perturbation of CYP1A1 by rs1048943 polymorphism causes an increase in the amount of harmful, adduct forming metabolic intermediates. A significant gene interaction between EPHX1 and NAT2 was also found (p=0.007). This interaction may affect the metabolism of harmful metabolic intermediates. Additionally, marginal interactions were found for other xenobiotic genes and these interactions may play a contributory role in clubfoot. Importantly, for CYP1A2, significant maternal (p=0.03; RR=1.24; 95% CI: 1.04-1.44) and fetal (p=0.01; RR=1.33; 95% CI: 1.13-1.54) genotypic effects were identified suggesting that both maternal and fetal genotypes impact normal limb development. No association was found for maternal smoking status and tobacco metabolism genes. Together, these results suggest that xenobiotic metabolism genes may play a contributory role in the etiology of clubfoot regardless of maternal smoking status and may impact foot development through perturbation of tobacco metabolic pathways.

Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene.

Persistently low white blood cell count (WBC) and neutrophil count is a well-described phenomenon in persons of African ancestry, whose etiology remains unknown. We recently used admixture mapping to identify an approximately 1-megabase region on chromosome 1, where ancestry status (African or European) almost entirely accounted for the difference in WBC between African Americans and European Americans. To identify the specific genetic change responsible for this association, we analyzed genotype and phenotype data from 6,005 African Americans from the Jackson Heart Study (JHS), the Health, Aging and Body Composition (Health ABC) Study, and the Atherosclerosis Risk in Communities (ARIC) Study. We demonstrate that the causal variant must be at least 91% different in frequency between West Africans and European Americans. An excellent candidate is the Duffy Null polymorphism (SNP rs2814778 at chromosome 1q23.2), which is the only polymorphism in the region known to be so differentiated in frequency and is already known to protect against Plasmodium vivax malaria. We confirm that rs2814778 is predictive of WBC and neutrophil count in African Americans above beyond the previously described admixture association (P = 3.8 x 10(-5)), establishing a novel phenotype for this genetic variant.

Multiple independent genetic factors at NOS1AP modulate the QT interval in a multi-ethnic population.

Extremes of electrocardiographic QT interval are associated with increased risk for sudden cardiac death (SCD); thus, identification and characterization of genetic variants that modulate QT interval may elucidate the underlying etiology of SCD. Previous studies have revealed an association between a common genetic variant in NOS1AP and QT interval in populations of European ancestry, but this finding has not been extended to other ethnic populations. We sought to characterize the effects of NOS1AP genetic variants on QT interval in the multi-ethnic population-based Dallas Heart Study (DHS, n = 3,072). The SNP most strongly associated with QT interval in previous samples of European ancestry, rs16847548, was the most strongly associated in White (P = 0.005) and Black (P = 3.6 x 10(-5)) participants, with the same direction of effect in Hispanics (P = 0.17), and further showed a significant SNP x sex-interaction (P = 0.03). A second SNP, rs16856785, uncorrelated with rs16847548, was also associated with QT interval in Blacks (P = 0.01), with qualitatively similar results in Whites and Hispanics. In a previously genotyped cohort of 14,107 White individuals drawn from the combined Atherosclerotic Risk in Communities (ARIC) and Cardiovascular Health Study (CHS) cohorts, we validated both the second locus at rs16856785 (P = 7.63 x 10(-8)), as well as the sex-interaction with rs16847548 (P = 8.68 x 10(-6)). These data extend the association of genetic variants in NOS1AP with QT interval to a Black population, with similar trends, though not statistically significant at P<0.05, in Hispanics. In addition, we identify a strong sex-interaction and the presence of a second independent site within NOS1AP associated with the QT interval. These results highlight the consistent and complex role of NOS1AP genetic variants in modulating QT interval.

Gene Discovery in Nonsyndromic Cleft Lip with or without Cleft Palate

Nonsyndromic cleft lip with or without cleft palate (NSCLP), a common, complex orofacial birth defect that affects approximately 4,000 newborns each year in the United States, is caused by both genetic and environmental factors. Orofacial clefts affect the mouth and nose, causing severe deformity of the face, which require medical, dental and speech therapies. Despite having substantial genetic liability, less than 25% of the genetic contribute to NSCLP has been identified. The studies described in this thesis were performed to identify genes that contribute to NSCLP and to demonstrate the role of these genes in normal craniofacial development. Using genome scan and candidate gene approaches, novel associations with NSCLP were identified. These include MYH9 (7 SNPs, 0.009≤p<0.05), Wnt3A (4 SNPs, 0.001≤p≤0.005), Wnt11 (2 SNPs, 0.001≤p≤0.01) and CRISPLD2 (4 SNPs, 0.001≤p<0.05). The most interesting findings were for CRISPLD2. This gene is expressed in the fused mouse palate at E17.5. In zebrafish, crispld2 localized to the craniofacial region by one day post fertilization. Morpholino knockdown of crispld2 resulted in a lower survival rates and altered neural crest cell (NCC) clustering. Because NCCs form the tissues that populate the craniofacies, this NCC abnormality resulted in cartilage abnormalities of the jaw including fewer ceratobranchial cartilages forming the lower jaw (three pairs compared to five) and broader craniofacies compared to wild-type zebrafish. These findings suggest that the CRISPLD2 gene plays an important role in normal craniofacial development and perturbation of this gene in humans contributes to orofacial clefting. Overall, these results are important because they contribute to our understanding of normal craniofacial development and orofacial clefting etiology, information that can be used to develop better methods to diagnose, counsel and potentially treat NSCLP patients.

Upregulation of defensins in burn sheep small intestine.

OBJECTIVE: The aim of this study was to visualize and localize the sheep antimicrobials, beta-defensins 1, 2, and 3, (SBD-1, SBD-2, SBD-3), sheep neutrophil defensin alpha (SNP-1), and the cathelicidin LL-37 in sheep small intestine after burn injury, our hypothesis being that these compounds would be upregulated in an effort to overcome a compromised endothelial lining. Response to burn injury includes the release of proinflammatory cytokines and systemic immune suppression that, if untreated, can progress to multiple organ failure and death, so protective mechanisms have to be initiated and implemented. METHODS: Tissue sections were probed with antibodies to the antimicrobials and then visualized with fluorescently labeled secondary antibodies and subjected to fluorescence deconvolution microscopy and image reconstruction. RESULTS: In both the sham and burn samples, all the aforementioned antimicrobials were seen in each of the layers of small intestine, the highest concentration being localized to the epithelium. SBD-2, SBD-3, and SNP-1 were upregulated in both enterocytes and Paneth cells, while SNP-1 and LL-37 showed increases in both the inner circular and outer longitudinal muscle layers of the muscularis externa following burn injury. Each of the defensins, except SBD-1, was also seen in between the muscle layers of the externa and while burn caused slight increases of SBD-2, SBD-3, and SNP-1 in this location, LL-37 content was significantly decreased. CONCLUSION: That while each of these human antimicrobials is present in multiple layers of sheep small intestine, SBD-2, SBD-3, SNP-1, and LL-37 are upregulated in the specific layers of the small intestine.

HLA-DPB1 and DPB2 are genetic loci for systemic sclerosis: a genome-wide association study in Koreans with replication in North Americans.

OBJECTIVE: To identify systemic sclerosis (SSc) susceptibility loci via a genome-wide association study. METHODS: A genome-wide association study was performed in 137 patients with SSc and 564 controls from Korea using the Affymetrix Human SNP Array 5.0. After fine-mapping studies, the results were replicated in 1,107 SSc patients and 2,747 controls from a US Caucasian population. RESULTS: The single-nucleotide polymorphisms (SNPs) (rs3128930, rs7763822, rs7764491, rs3117230, and rs3128965) of HLA-DPB1 and DPB2 on chromosome 6 formed a distinctive peak with log P values for association with SSc susceptibility (P=8.16x10(-13)). Subtyping analysis of HLA-DPB1 showed that DPB1*1301 (P=7.61x10(-8)) and DPB1*0901 (P=2.55x10(-5)) were the subtypes most susceptible to SSc in Korean subjects. In US Caucasians, 2 pairs of SNPs, rs7763822/rs7764491 and rs3117230/rs3128965, showed strong association with SSc patients who had either circulating anti-DNA topoisomerase I (P=7.58x10(-17)/4.84x10(-16)) or anticentromere autoantibodies (P=1.12x10(-3)/3.2x10(-5)), respectively. CONCLUSION: The results of our genome-wide association study in Korean subjects indicate that the region of HLA-DPB1 and DPB2 contains the loci most susceptible to SSc in a Korean population. The confirmatory studies in US Caucasians indicate that specific SNPs of HLA-DPB1 and/or DPB2 are strongly associated with US Caucasian patients with SSc who are positive for anti-DNA topoisomerase I or anticentromere autoantibodies.

Altered transmission of HOX and apoptotic SNPs identify a potential common pathway for clubfoot.

Clubfoot is a common birth defect that affects 135,000 newborns each year worldwide. It is characterized by equinus deformity of one or both feet and hypoplastic calf muscles. Despite numerous study approaches, the cause(s) remains poorly understood although a multifactorial etiology is generally accepted. We considered the HOXA and HOXD gene clusters and insulin-like growth factor binding protein 3 (IGFBP3) as candidate genes because of their important roles in limb and muscle morphogenesis. Twenty SNPs from the HOXA and HOXD gene clusters and 12 SNPs in IGFBP3 were genotyped in a sample composed of non-Hispanic white and Hispanic multiplex and simplex families (discovery samples) and a second sample of non-Hispanic white simplex trios (validation sample). Four SNPs (rs6668, rs2428431, rs3801776, and rs3779456) in the HOXA cluster demonstrated altered transmission in the discovery sample, but only rs3801776, located in the HOXA basal promoter region, showed altered transmission in both the discovery and validation samples (P = 0.004 and 0.028). Interestingly, HOXA9 is expressed in muscle during development. An SNP in IGFBP3, rs13223993, also showed altered transmission (P = 0.003) in the discovery sample. Gene-gene interactions were identified between variants in HOXA, HOXD, and IGFBP3 and with previously associated SNPs in mitochondrial-mediated apoptotic genes. The most significant interactions were found between CASP3 SNPS and variants in HOXA, HOXD, and IGFBP3. These results suggest a biologic model for clubfoot in which perturbation of HOX and apoptotic genes together affect muscle and limb development, which may cause the downstream failure of limb rotation into a plantar grade position.

Prognostic significance of xCT polymorphisms and expression in patients with advanced pancreatic cancer treated with chemotherapy

The plasma membrane xc- cystine/glutamate transporter mediates cellular uptake of cystine in exchange for intracellular glutamate and is highly expressed by pancreatic cancer cells. The xCT gene, encoding the cystine-specific xCT protein subunit of xc-, is important in regulating intracellular glutathione (GSH) levels, critical for cancer cell protection against oxidative stress, tumor growth and resistance to chemotherapeutic agents including platinum. We examined 4 single nucleotide polymorphisms (SNPs) of the xCT gene in 269 advanced pancreatic cancer patients who received first line gemcitabine with or without cisplatin or oxaliplatin. Genotyping was performed using Taqman real-time PCR assays. A statistically significant correlation was noted between the 3' untranslated region (UTR) xCT SNP rs7674870 and overall survival (OS): Median survival time (MST) was 10.9 and 13.6 months, respectively, for the TT and TC/CC genotypes (p = 0.027). Stratified analysis showed the genotype effect was significant in patients receiving gemcitabine in combination with platinum therapy (n = 145): MST was 10.5 versus 14.1 months for the TT and TC/CC genotypes, respectively (p = 0.013). The 3' UTR xCT SNP rs7674870 may correlate with OS in pancreatic cancer patients receiving gemcitabine and platinum combination therapy. Paraffin-embedded core and surgical biopsy tumor specimens from 98 patients with metastatic pancreatic adenocarcinoma were analyzed by immunohistochemistry using an xCT specific antibody. xCT protein IHC expression scores were analyzed in relation to overall survival in 86 patients and genotype in 12 patients and no statistically significant association was found between the level of xCT IHC expression score and overall survival (p = 0.514). When xCT expression was analyzed in terms of treatment response, no statistically significant associations could be determined (p = 0.908). These data suggest that polymorphic variants of xCT may have predictive value, and that the xc- transporter may represent an important target for therapy in pancreatic cancer.

Theoretical and experimental studies of linkage disequilibrium in human populations

Linkage disequilibrium (LD) is defined as the nonrandom association of alleles at two or more loci in a population and may be a useful tool in a diverse array of applications including disease gene mapping, elucidating the demographic history of populations, and testing hypotheses of human evolution. However, the successful application of LD-based approaches to pertinent genetic questions is hampered by a lack of understanding about the forces that mediate the genome-wide distribution of LD within and between human populations. Delineating the genomic patterns of LD is a complex task that will require interdisciplinary research that transcends traditional scientific boundaries. The research presented in this dissertation is predicated upon the need for interdisciplinary studies and both theoretical and experimental projects were pursued. In the theoretical studies, I have investigated the effect of genotyping errors and SNP identification strategies on estimates of LD. The primary importance of these two chapters is that they provide important insights and guidance for the design of future empirical LD studies. Furthermore, I analyzed the allele frequency distribution of 26,530 single nucleotide polymorphisms (SNPs) in three populations and generated the first-generation natural selection map of the human genome, which will be an important resource for explaining and understanding genomic patterns of LD. Finally, in the experimental study, I describe a novel and simple, low-cost, and high-throughput SNP genotyping method. The theoretical analyses and experimental tools developed in this dissertation will facilitate a more complete understanding of patterns of LD in human populations. ^

CCTeta: A novel soluble guanylyl cyclase-interacting protein

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of 3′,5 ′-cyclic guanosine monophosphate (cGMP) from 5′ -guanosine triphosphate (GTP). In this thesis, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit η and the α1β1 isoform of sGC. Using the yeast-two-hybrid system, CCTη was found to interact with the N-terminal portion of β1 subunit of sGC. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast-two-hybrid system, CCTη was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTη and Sf9 lysate expressing sGC resulted in a 33% inhibition of sodium nitroprusside (SNP)-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTη had no effect on this activity. Furthermore, CCTη had no effect on the activity of αβCys105 sGC a constitutively active mutant that lacks a heme group. Of note is the fact that the full-length CCTη-expressing bacterial lysate inhibited the activity of sGC-expressing Sf9 lysate by 48% compared with GST alone. This indicates that the amino terminal 94 amino acids of CCTη are important to the inhibition of sGC activity. Lastly, a 45% inhibition of sGC activity by CCTη was seen in vivo in BE2 cells stably transfected with CCTη and treated with SNP. The fact that the inhibition of sGC was more pronounced with bacterial lysate expressing CCTη versus the purified CCTη implies that some factor in the bacterial lysate enhances the inhibitory effect of CCTη. Because the level of inhibition seen in bacterial lysate and in vivo experiments is similar, might imply that the factor that aids in CCTη effect on sGC is conserved. Together, these data suggest that CCTη is a novel type of sGC inhibitor that inhibits sGC by modifying the binding of NO to the heme group or the subsequent conformational changes induced by NO binding. ^

DNA nucleotide excision repair gene single nucleotide polymorphisms and hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease caused by germline mutations in DNA mismatch repair(MMR) genes. The nucleotide excision repair(NER) pathway plays a very important role in cancer development. We systematically studied interactions between NER and MMR genes to identify NER gene single nucleotide polymorphism (SNP) risk factors that modify the effect of MMR mutations on risk for cancer in HNPCC. We analyzed data from polymorphisms in 10 NER genes that had been genotyped in HNPCC patients that carry MSH2 and MLH1 gene mutations. The influence of the NER gene SNPs on time to onset of colorectal cancer (CRC) was assessed using survival analysis and a semiparametric proportional hazard model. We found the median age of onset for CRC among MMR mutation carriers with the ERCC1 mutation was 3.9 years earlier than patients with wildtype ERCC1(median 47.7 vs 51.6, log-rank test p=0.035). The influence of Rad23B A249V SNP on age of onset of HNPCC is age dependent (likelihood ratio test p=0.0056). Interestingly, using the likelihood ratio test, we also found evidence of genetic interactions between the MMR gene mutations and SNPs in ERCC1 gene(C8092A) and XPG/ERCC5 gene(D1104H) with p-values of 0.004 and 0.042, respectively. An assessment using tree structured survival analysis (TSSA) showed distinct gene interactions in MLH1 mutation carriers and MSH2 mutation carriers. ERCC1 SNP genotypes greatly modified the age onset of HNPCC in MSH2 mutation carriers, while no effect was detected in MLH1 mutation carriers. Given the NER genes in this study play different roles in NER pathway, they may have distinct influences on the development of HNPCC. The findings of this study are very important for elucidation of the molecular mechanism of colon cancer development and for understanding why some mutation carriers of the MSH2 and MLH1 gene develop CRC early and others never develop CRC. Overall, the findings also have important implications for the development of early detection strategies and prevention as well as understanding the mechanism of colorectal carcinogenesis in HNPCC. ^

Characterization of glucocorticoid receptor (NR3C1) gene polymorphisms and a family-based association study with hypertension

Hypertension is usually defined as having values of systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg. Hypertension is one of the main adverse effects of glucocorticoid on the cardiovascular system. Glucocorticoids are essential hormones, secreted from adrenal glands in circadian fashion. Glucocorticoid's effect on blood pressure is conveyed by the glucocorticoid receptor (NR3C1), an omnipresent nuclear transcription factor. Although polymorphisms in this gene have long been implicated to be a causal factor for cardiovascular diseases such as hypertension, no study has yet thoroughly interrogated the gene's polymorphisms for their effect on blood pressure levels. Therefore, I have first resequenced ∼30 kb of the gene, encompassing all exons, promoter regions, 5'/3' UTRs as well as at least 1.5 kb of the gene's flanking regions from 114 chromosome 5 monosomic cell lines, comprised of three major American ethnic groups—European American, African American and Mexican American. I observed 115 polymorphisms and 14 common molecularly phased haplotypes. A subset of markers was chosen for genotyping study populations of GENOA (Genetic Epidemiology Network of Atherosclerosis; 1022 non-Hispanic whites, 1228 African Americans and 954 Mexican Americans). Since these study populations include sibships, the family-based association test was performed on 4 blood pressure-related quantitative variables—pulse, systolic blood pressure, diastolic blood pressure and mean arterial pressure. Using these analyses, multiple correlated SNPs are significantly protective against high systolic blood pressure in non-Hispanic whites, which includes rsb198, a SNP formerly associated with beneficial body compositions. Haplotype association analysis also supports this finding and all p-values remained significant after permutation tests. I therefore conclude that multiple correlated SNPs on the gene may confer protection against high blood pressure in non-Hispanic whites. ^