Sickle cell disease is the most common monogenic genetic disease in the United States. Mortality in sickle cell disease is higher in those with elevated pulmonary pressures or other markers of disease severity including frequent pain, iron overload and renal insufficiency. We have enrolled 716 subjects and 115 controls in a study of the prevalence and prognosis of subjects with sickle cell disease and suspected pulmonary hypertension. This represents approximately a 1% sample of patients with sickle cell disease living in the United States. All subjects were screened with transthoracic echocardiograms and the tricuspid regurgitant jet velocity (TRV) used to estimate the pulmonary artery systolic pressure. Additional phenotypic information related to the manifestations of sickle cell disease is also collected at evaluation. Suspected pulmonary hypertension was prospectively defined by a TRV >= 2.5 m/sec and severe pulmonary hypertension defined by a TRV >= 3.0 m/sec. Subjects have been followed for a mean of 6 years and censored at time of death or loss to follow-up. There were 69 new subjects enrolled at the NIH during this year;10 were controls and 59 were subjects with sickle cell disease. There were no new subjects enrolled at Howard University. Total enrolled at Howard University is 131 and the total enrolled at NIH is 700. Total enrollment for all sites is 831. Subject enrollment is ongoing. We are conducting 2, 4, 6, 8, 10 and 12 year follow-up visits for comprehensive data collection. We also completing a new comprehensive survival update on all subjects. All new subjects had corresponding biospecimens collected for genomic investigations. Our goal is to recruit at least 1000 subjects with sickle cell disease for initial exploratory genetic studies. This will allow for sufficient statistical power to preliminarily identify genetic modifiers. Variants in the GCH1 gene, which have previously been identified as modulators of pain in other diseases, are associated with increased risk of severe vaso-occlusive pain crises in a case control study. We also replicated these findings in an independent cohort. GCH1 is the rate limiting step for the synthesis of BH4 (tetrahydrobiopterin) which is an essential co-factor for the synthesis of nitric oxide, catecholamines, serotonin, and phenylalanine metabolism. In vitro analysis of whole blood samples from subjects with sickle cell disease shows that this GCH1 haplotype is associated with higher BH4 levels. We believe that this result indicates that this is a unique African haplotype associated with increased BH4 and increased pain. This haplotype is also associated with altered vascular function during forearm blood flow studies in adults with sickle cell disease. Surprisingly, this haplotype is not associated with pulmonary hypertension in SCD despite the biological suggestion that the African haplotype might promote nitric oxide production. Variants in the MYH9 gene are associated with decreased glomerular filtration rate in SCD. Genetic markers in the region of MYH9 on chromosome 22 have been identified as major risk factors for focal segmental glomerular sclerosis (FSGS) in African American populations. This candidate gene association study extends this work to a disease specific patient population that is at high risk for renal insufficiency. A genome wide association study several phenotypes in sickle cell anemia is in progress using ancestry informative markers that were identified from the HapMap project. We have designed a genome wide panel of 1806 ancestry informative markers and have completed genotyping in more than 1700 subjects with sickle cell disease and 2 HapMap study populations. Analysis of population structure using this panel demonstrates that a proportion of sickle cell disease patients in the United States have very little genetic admixture. The degree of population admixture is also significantly different when comparing sickle cell disease populations from different geographic regions of the United States. These results suggest that genetic studies of sickle cell disease may require additional statistical adjustments when combining cohorts. The highly variable degree of admixture also implies that statistical power may be significantly lower than current predictions. For genome wide association studies, we will perform a combined analysis with samples from the Bethesda Sickle Cell Cohort Study ( Identifier NCT00011648) and the WalkPhasst clinical trial screening population ( Identifier NCT00492531). Initially, we will analyze pain and HbF as phenotypes. The phenotype defining cases in this study is >1 severe pain crisis per year which provides >80% statistical power to detect genome wide associations with a relative risk of 2 or higher. Studies are also underway to also determine if gene expression profiling can also be used to identify sub-phenotypes of sickle cell disease. In a pilot study, we have isolated RNA from different peripheral blood preparations to determine which is the most informative for larger studies. For this pilot, subjects with SS sickle cell disease were compared to those with SC sickle cell disease because they each have distinct clinical manifestations. Preliminary analysis suggests that SS and SC have nearly identical gene expression profiles despite their clinical differences. In a larger follow-up study, we compared gene expression profiles for 2 sickle cell anemia sub-phenotypes defined by a plasma biomarker using exon arrays. We have identified candidate genes associated with this sub-phenotype, including several alternatively spliced transcripts.

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Belfer, Inna; Youngblood, Victoria; Darbari, Deepika S et al. (2014) A GCH1 haplotype confers sex-specific susceptibility to pain crises and altered endothelial function in adults with sickle cell anemia. Am J Hematol 89:187-93
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