Genome-Wide Association Study for Hb F Determinants in Sardinian Patients with Thalassemia Major and Intermedia.

Expression of fetal globin is silenced normally in adult life; however, determinants linked and/or unlinked to the globin-gene clusters could modify Hb F expression so it persists into adults. Increased expression in adults offers hope as a cure for sickle cell disease (SCD) and beta thalassemia, since formation of FS hybrids in SCD inhibits deoxy Hb S polymerization while increased fetal chain expression compensates partially for decreased adult beta-globin chains in beta thalassemia. Characterization and controlled manipulation of high Hb F determinants is critical to decreasing clinical severity of these life-threatening genetic diseases, which result in high morbidity and mortality worldwide. We report on analysis of a unique beta-thalassemia cohort from Sardinia who present with either

1. a mild, non-transfusion-dependent (NTD) form expressing high Hb F, or with

2. a severe, transfusion-dependent (TD) form expressing low Hb F.

Both groups are homozygous for the beta39 chain-termination mutation and lack adult beta globin. Genome-wide DNA arrays were run on 42 TD and 33 NTD patients using the Affymetrix 500K (500,568 SNPs in a total of 23 individuals) or 6.0 (909,622 SNPs in a total of 52 individuals) SNP chip platforms. The average sample call rates were 96.3% for the 6.0 chip and 94.3% for the 500K chip. Data from the two chips were merged and a total of 482,243 SNPs that were present in both chips were considered for genetic association analysis using a case/control design (NTD versus TD). Quality control filters removed 42,166 SNPs (8.7%) with genotype call rates < 90%. An additional 54,683 SNPs (12.4%) were removed because they either failed a test of Hardy-Weinberg equilibrium at a p< 0.0001 or had a minor allele frequency (MAF) of < 0.01. The genotype call rate for the remaining 385,394 SNPs was 97.3%. Preliminary analysis of difference in allele frequency distributions in the two groups of patients did not reveal any signal of association that remained statistically significant when corrected for multiple tests. The smallest p-value observed was 2x10-6 for SNP rs8028098 on chromosome 15. However, several SNPs in two candidate regions on chromosomes 2p16 and 6q23 were nominally significant (p<0.05). The smallest p-values observed in these two regions were 0.004 for rs11886868 located in the BCL11A gene, and 0.002 for rs210950 in the MYB gene. These results confirm the likely involvement of these two genes in the regulation of Hb F expression, and show the usefulness of the association approach in the search for genetic modifiers of Mendelian disease phenotypes. Additional samples are being analyzed in an attempt to achieve sufficient power to reach genome-wide significance.