High Frequency of Large Gene Deletions Detected by Multiplex Ligation-Dependent Probe Amplification in Diamond Blackfan Anemia,

Abstract 3428 Background: Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Although mutations of eleven ribosomal protein (RP) genes have been detected in more than 50% of DBA patients the remaining patients appear to have intact ribosomal protein genes using stadard sequencing methods (Boria et al. Hum Mut 2010). We previously described the detection of three large RPS19 deletions using the MLPA (Multiplex Ligation-dependent Probe Amplification) technique (Quarello et al. Haematologica 2008). As MLPA is an efficient and rapid technique that detects gene dosage alterations, we thus decided to apply this approach also to other RP genes. Aim: To search for unidentified RP large deletions we applied the MLPA technique in Italian DBA patients who have been found mutation-negative by sequencing. Methods: Italian DBA patients without RP genes mutations (73/156, 47%) were included in this study. The analysis was performed using a homemade MLPA kit following the recommendations provided by MRC Holland (Amsterdam, The Netherlands, www.mlpa.com). The probes were designed to detect deletions of six RP genes (RPS17, RPS19, RPS26, RPL5, RPL11, RPL35A). Deletions of probe recognition sequences were apparent by a 35–50% reduced relative peak area of the amplification product of that probe. Results: The results of the MLPA assay revealed that 13 out of the 73 probands (18%) had a multi-exonic deletion in one of the six DBA genes analyzed. We identified four deletions of the RPS17 gene, three of the RPS26 gene, three of the RPL35A, two of the RPL11 gene and one of the RPL5 gene. No additional RPS19 deletions were found. DBA patients with deletions showed a severe phenotype with a very high percentage of transfusion dependence (85%). Somatic malformations were observed only in two patients. Conclusion: We detected a high percentage of deletions of known DBA genes in a cohort of patients in whom no mutations were found by RP sequencing. Mutation screening of the RP genes with a combination of sequencing and MLPA reached an overall detection rate of 61.5% (96/156). In our cohort, large genomic deletions represent up to 18% of all mutations detected. In conclusion, we stress the high percentage of identified RP genes deletions in DBA patients. We also highlight that a gene-dosage technique, such as MLPA, should complement sequencing in a clinical environment since only a combined approach of this kind permits the comprehensive detection of all mutations in the DBA RP genes. Disclosures: No relevant conflicts of interest to declare.