Intravascular hemolysis in sickle cell anemia patients produces increased circulating heme levels and has been associated with clinical complications including cholestatic jaundice and cholelithiasis 
Unconjugated hyperbilirubinemia is a risk factor for the development of bile pigment stones in sickle cell disease and other hemolytic anemias 
. Typically, bilirubin is formed from metabolism of heme, which is derived principally from erythrocytic hemoglobin. Heme is oxidatively metabolized by heme oxygenase-1 into biliverdin, which is subsequently reduced enzymatically by biliverdin reductase to bilirubin. Upon formation, bilirubin is water insoluble, due to the presence of internal hydrogen bonding that creates a contorted molecule structure, inhibiting solubility. This form of bilirubin, termed unconjugated or indirect bilirubin, is responsible for all of the toxic effects of bilirubin observed clinically. To permit excretion, bilirubin complexes with albumin and is transported via the bloodstream to the liver, where it undergoes glucuronidation by the UGT family of enzymes; UGT1A1
is the only one with clinical relevance in humans 
is a gene complex composed of 9 transcriptional units encoding an isoform of the UGT
. The most common genetic cause of impaired bilirubin glucuronidation occurs in Gilbert’s syndrome, which follows an autosomal recessive inheritance pattern and most commonly affects Caucasians of European descent 
. Clinically, these patients have a mild indirect hyperbilirubinemia, which worsens during periods of stress or febrile illnesses 
. Genetic studies of these patients have identified a dinucleotide repeat polymorphism (TA)5–8
in the TATA box of the UGT1A1
gene promoter that is associated with reduced UGT
expression and produces hyperbilirubinemia 
. This polymorphism has also been observed in small cohorts of sickle cell anemia patients suggesting a common pathogenic link between ethnically divergent etiologies of indirect hyperbilirubinemia 
. In the current study, genome-wide genetic screening of a large cohort of sickle cell anemia patients not only confirmed these findings but also supported their biological relevance.
In addition to defects in bilirubin metabolism, genetic alterations in the glucuronidation pathway have been linked to abnormalities in the hepatic metabolism of certain medications, pre-disposition to cardiovascular disease and certain types of malignancy suggesting a clinical level of importance beyond cholelithiasis 
. How this relates to the pathogenesis of sickle cell anemia is not entirely clear clinically, but may be worthy of future study.
In genetic studies of primarily Caucasian cohorts, cholelithiasis and serum bilirubin levels are heritable traits. Family-based studies show that the UGT
locus accounted for a significant proportion of the variation observed in both of these variables 
. Our study is the first to use GWAS to examine an African American population with sickle cell anemia, a condition where hemolysis predisposes patients to hyperbilirubinemia, to determine the heritability of serum bilirubin levels. Fifteen SNPs in UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8
reached genome-wide significance for association with total bilirubin levels in the CSSCD cohort; 13 of these were also associated with the presence or history of cholelithiasis. These findings were confirmed in 4 other cohorts totaling 3,269 patients, unusual for a rare disease such as sickle cell anemia, and representing a greater number of patients than all of the previous studies of this population combined.
SNPs we identified in the current study have been linked to hyperbilirubinemia in other populations. SNP rs887829, the most significant SNP associated with bilirubin levels identified in the CSSCD, Duke and Walk-PHaSST cohorts, was previously observed in associated with total bilirubin levels in a GWAS of 4,300 Sardinians 
. This study also reported a significant association with bilirubin and the SLCO1B3
locus; however, we were unable to replicate these results in our study due to unavailability or low MAF of the markers in our array. In a study performed by Cheng et al. examining genetic variants associated with serum bilirubin in 619 healthy African Americans revealed that the top SNP was rs887829 (p
. This suggests that this variant is not unique to African Americans with sickle cell disease. It has been theorized that this SNP may confer protection against malaria which may explain its penetrance amongst African populations. SNP rs887829 is located in the promoter region of UGT1A1
, 221 bp upstream from the (TA)n
repeat sequence. Horsfall et al. found a strong association was found between rs887829 and the (TA)7
repeat sequence suggesting that this may be a marker for the (TA)7/(TA)7 or 8 genotypes 
. This proximity to the promoter element and strong level of association suggest that rs887829 is a marker for the (TA)n
repeats and that additional sequencing of this gene would be redundant 
. SNP rs6742078, identified in the current study, was associated with total bilirubin in a meta-analysis previously performed by Johnson et al., including the Framingham Heart Study, Rotterdam Study and Age, Gene, Environment and Susceptibility-Reykjavik study cohorts 
. In addition, SNPs rs3755319, rs7586110, and rs6759892 were also found to be significantly associated with total bilirubin in a cohort of 4,300 Sardinians 
. SNP rs887829 was found to be associated with cholelithiasis in a study published by Buch et al., in which a cohort of 2,606 German cholelithiasis patients was compared with 1,121 South American controls (OR
1.73, p value .003) 
There was no association between LDH, hemoglobin concentration or reticulocyte count, markers of hemolysis, and the SNPs identified in the present study. A potential explanation for this is the weak association between bilirubin levels and hemolytic rate. While the "gold standard" for hemolysis is the red cell lifespan, this is rarely performed and surrogate blood biomarkers such as reticulocyte count, LDH, bilirubin and haptoglobin levels are used clinically to estimate the degree of hemolysis. None of these measures are specific for hemolysis. An elevated serum bilirubin level in sickle cell anemia reflects multiple pathophysiologic processes that include liver disease in addition to hemolysis and therefore may lack the phenotypic specificity required for genetic association studies of hemolysis. Another possible reason for this finding is that the genes associated with bilirubin levels are all involved in bilirubin catabolism and not production suggesting that they are not reflective of the processes leading to bilirubin formation.
In summary, SNPs in UGT1A1 are most tightly associated with bilirubin levels in African Americans with sickle cell anemia. This study in conjunction with those conducted in other populations at risk for unconjugated hyperbilirubinemia and other cohorts of sickle cell anemia patients support the concept that genetically mediated differences in bilirubin conjugation play an important role in the cholelithiasis risk. It is possible that targeting this pathway pharmacologically may offer new therapeutic options for these patients.