A Genome-Wide Association Study of Total Bilirubin and Cholelithiasis Risk in Sickle Cell Anemia

Serum bilirubin levels have been associated with polymorphisms in the UGT1A1 promoter in normal populations and in patients with hemolytic anemias, including sickle cell anemia. When hemolysis occurs circulating heme increases, leading to elevated bilirubin levels and an increased incidence of cholelithiasis. We performed the first genome-wide association study (GWAS) of bilirubin levels and cholelithiasis risk in a discovery cohort of 1,117 sickle cell anemia patients. We found 15 single nucleotide polymorphisms (SNPs) associated with total bilirubin levels at the genome-wide significance level (p value <5×10−8). SNPs in UGT1A1, UGT1A3, UGT1A6, UGT1A8 and UGT1A10, different isoforms within the UGT1A locus, were identified (most significant rs887829, p = 9.08×10−25). All of these associations were validated in 4 independent sets of sickle cell anemia patients. We tested the association of the 15 SNPs with cholelithiasis in the discovery cohort and found a significant association (most significant p value 1.15×10−4). These results confirm that the UGT1A region is the major regulator of bilirubin metabolism in African Americans with sickle cell anemia, similar to what is observed in other ethnicities

Manhattan plot showing the association of SNPs with systolic blood pressure (SBP).

<p>The genome-wide distribution of -log₁₀ P-values are plotted against the physical position of each SNP on each chromosome. The threshold for genome-wide significance (P-value < 5.0x10^-8) is indicated by the horizontal dashed line.</p

Single Nucleotide Polymorphisms Associated with Total Bilirubin Levels.

<p>Genome wide significant SNPs in the CSSCD study and their replicates in the independent cohorts. The table reports the SNP identifier from dbSNP, chromosome, physical coordinates (hg18), the coded allele in PLINK (also minor allele) and the non-coded allele, the minor allele frequency (MAF) from the CSSCD cohort, the gene clusters where the SNP is located, and regression coefficientt and p-value in each study. Additive models of association were used in all studies. NA in the MSH means the SNP was unavailable in the 370 Illumina array.</p

Single Nucleotide Polymorphisms Associated with Cholelithiasis.

<p>Results of SNP association analysis with cholelithiasis in the CSSCD study using the additive model. The minor allele is the coded allele, and the OR is the odds for cholelithiasis in carriers of one extra copy of the coded allele.</p

Patient Characteristics in CSSCD, MSH and Walk-PHaSST cohorts.

*<p>Walk-PHaSST bilirubin measurement is in SI units.</p><p>Summary statistics of patient characteristics in the CSSCD, MSH, and WALK-PHaSSTstudies. For each study, the first column reports statistics (mean and standard deviation) for all patients included in the analysis and the second and third columns report statistics stratified by gender.</p

LD Structure in the CSSCD Cohort.

<p>LD plots for regions in genes <i>UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9</i> and <i>UGT1A10</i> on chromosome 2 in the CSSCD subjects. The LD plot was generated using Haploview 4.2. Each diamond represents the D’ value between two SNPs. The LD color scheme is: white D’<1 and LOD<2, blue D’ = 1 and LOD<2; shades of pinkish-red D’<1 and LOD≥2 and bright red D’ = 1 and LOD≥2.</p

Association Analysis with LDH, Reticulocyte Counts and Hemoglobin Concentration.

<p>Results of SNP association analysis with other hemolytic phenotypes including hemoglobin, LDH and reticulocyte count in the CSSCD Study.</p

Summary of the GWAS data from the CSSCD Cohort.

<p>The Manhattan plot (A) displays the –log10(p value) of the associations tested in the CSSCD cohort using the additive model. Color bands represent chromosomes, and SNPs are ordered by their physical position within each chromosome. The large spike in chromosome 2 corresponds to the <i>UGT1A1, UGT1A3, UGT1A8</i> and <i>UGT1A10</i> regions. The QQ-plot (B) displays the observed (y-axis) versus expected (x-axis) –log10 (p-value). From the QQ plot, there is minimal to no inflation in the test statistic.</p