High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nThe finding of partial or complete RP deletions/duplications in DBA patients using CGH array, SNP array or quantitative PCR has been recently reported. [13][14][15] In order to better characterize the molecular defect in our cohort of Italian DBA patients who were negative for RP gene mutations by standard sequencing, we developed an MLPA synthetic probe set that allows the concurrent analysis of the six principal RP genes: RPS19, RPL5, RPL11, RPL35A, RPS17 and RPS26. Design and Methods PatientsPatients were selected from the Italian DBA Registry that includes 173 Italian subjects from 162 families. Diagnosis was reached following the criteria suggested by the DBA International Clinical Consensus (ICC) Consortium.2 All the patients were screened for mutations in ten genes.RPS19 mutations were found in 44 of 162 (27%) unrelated DBA patients using a strategy that included sequencing and the MLPA technique. The sequencing analysis of RPS24, RPS26, RPL5, and RPL11, revealed mutations in 46 of 162 (28%) unrelated patients. No mutations were found in RPS10, RPS14, RPS16, RPS17, or RPL35A. Seventy-two (45%) unrelated DBA patients showed no mutations in these genes and were included in this study. Informed consent was obtained from all patients and/or their legal guardians. MLPA design and methodMLPA oligonucleotide probes were designed according to the "Designing synthetic MLPA probes" protocol v.8 (MRC-Holland, Amsterdam, The Netherlands) that uses 12 probes per assay. Each synthetic probe was composed of two 5′ and 3′ half-probes containing unique target-specific sequence and universal primer sequences 5′-GGGTTCCCTAAGGGTTGGA and 5′-TCTA-GATTGGATCTTGCTGGCAC on their 5′ and 3′ ends, respectively. Primers were designed in regions that did not include single nucleotide polymorphisms (SNPs) (http://genome.ucsc.edu/).Because of the constraint of including 12 probes, we were forced to choose a strategy aimed at the screening of whole gene deletions or duplications. The probemix contained probes interrogating 1-3 exons per each RP gene (Online Supplementary Table S1, Figure 1A and B).MLPA was performed on genomic DNA according to the manufacturer's instructions. We used the P200-A1 Human DNA reference kit that includes reference probes and MLPA control fragments (http://www.mrc-holland.com).The PCR products were diluted 1:15 in water, 1 mL of this solution was mixed with 10 mL of HiDi formamide (ABI) and 0.2 mL of ROX500 size standard. The amplification products were separated by capillary electrophoresis on an ABI 3100 Avant Genetic Analyzer (Applied Biosystems, Warrington, UK). Genescan v3.7 software was used to analyze the runs and to retrieve peak intensities corresponding to each probe in the different samples and the integrated peak areas were exported to an Excel 2003 spreadsheet (Microsoft, CA, USA).Normalization was obtained by dividing the peak area of each probe's amplification product by the combined peak areas of all probes (intra-normalization). We then ...

Genomic analysis of bone marrow failure and myelodysplastic syndromes reveals phenotypic and diagnostic complexity

“…42 The deep coverage afforded by this targeted sequencing approach detected CNVs with a single assay, in contrast to alternative approaches combining targeted capture, array comparative genomic hybridization (aCGH), and RNA sequencing. 43 This is especially important for inherited BMF/MDS which are frequently caused by deletions in ribosomal protein genes, 44,45 RUNX1, 28,46 and FANCA. 47,48 Finally, whole exome sequencing queries thousands of genes unrelated to BMF/MDS and thus may reveal incidental mutations not immediately relevant to the diagnosis that prompted the sequencing.…”

Marrow failure: a window into ribosome biology

“…Of these ribosomal gene mutations, around 20% involve large deletions that require analysis of copy number variation for detection. 13 Ribosomal gene mutations may be inherited in an autosomal dominant pattern or may arise spontaneously. X-linked mutations in the transcription factor GATA1, which is essential for erythropoiesis, have also been associated with DBA.…”

The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70

“…[8][9][10][11] DBA was the first identified human ribosomopathy. 12 A constitutive heterozygous mutation, including large deleterious deletions, [13][14][15] in 14 ribosomal protein (RP) genes 13,[16][17][18][19][20][21][22][23] is identified in 70% of DBA cases. The disease is characterized by an erythroblastopenia in an otherwise normocellular and nondysplastic bone marrow.…”