Searching for Disease Modifiers Genes in Thalassemia.

β0-thalassemia/HbE disease is one of the most common thalassemias in Southeast Asia. Patients with compound heterozygote for HbE and β0 mutant alleles display remarkable variability in clinical expression, ranging from nearly asymptomatic to severe, transfusion-dependent disease. It is believed that additional genetic factors modifying disease severity may account for this variability. A universal platform technology (MassARRAY) based on mass spectrometry for analyzing nucleic acids at a high level of precision and accuracy has been developed. To identify genetic modifiers influencing severity among 1060 β0-thalassemia/HbE patients, we are utilizing this technology to conduct a genome-wide association study involving approximately 110,000 gene-based single nucleotide polymorphisms (SNPs). This assay panel corresponds to SNPs with a median spacing of 10.4 kilobases, in approximately 99% of all known and predicted human genes. DNAs from approximately 200 regionally matched patients representing the extremes of mild and severe cases were included in each DNA pool. Allele frequencies for all SNPs were estimated in both pools, and those showing suggestive significant differences (p values <0.02) were selected for verification by repeated pooled DNA analysis. Approximately one-fourth of these showed reproducible allelic differences at p<0.05, and more than 600 were selected for genotyping the individual patient DNAs in order to determine precise allele and genotype frequencies. A number of SNPs showed evidence for association with disease severity, including several in reported quantitative trait loci (QTLs) associated with fetal hemoglobin HbF levels. However the most strongly associated SNPs were within a region on chromosome 11 distinct from the beta globin gene cluster, within which most analysis to date has focused.