Analysis of Rare deletional Thalassemia Using Custom CGH Array DNA Chip

Abstract 4278

Deletions are the main etiology for alpha thalassemia and account for 9 to 10 % of beta thalassemia. They are currently detected using “in house” methods as QMPSF or the use of commercially available assays as MLPA. The accurate determination of the breakpoint could be of importance for news and rare events to have a proper characterization of the deletion and to understand the consequences of the event. However, the imprecision due to the relative small numbers of markers in these methods, the distance to be covered and the high percentage of repeated sequences located into the intergenic regions, makes the breakpoint determination difficult. To overcome the limitation of the number of markers we have setup a custom “Comparative Genomic Hybridization” assay to study the beta and the alpha globin cluster. Based upon the Agilent multiple arrays system, we have designed an array covering chromosome 11 and chromosome 16, containing 15 000 probes with an average spacing of 150 bp on both clusters. This CGH DNA chip was used to successfully clone the breakpoint of several rare and new deletions removing from 10 kb to up to 1 megabase of DNA sequence on chromosome 11 and chromosome 16. The results show the efficiency of such an approach and the multiple array format allows a good cost value by comparison to step by step cloning. In 50 % from more than 100 beta locus deletional events the 5′ breakpoint lies between the Aγ globin and the delta globin gene indicating the location of hotspots of recombination herein. Recent findings have shown that intergenic sequences in β-globin cluster and probably in α-globin cluster may be involved in the global regulation of the globin genes expression. Interestingly, a non coding small RNA was recently discovered in the same region and supposed to be involved in fetal hemoglobin expression. Such multiple arrays CGH chips provide an easy and efficient tool to define critical intergenic regions involved in the global regulation of the globin genes. Such hypothesis is currently under study.