Novel deletion of RPL15 identified by array-comparative genomic hybridization in Diamond-Blackfan anemia

Abstract

Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30-50 % of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26 and GATA1 in about 60-65 % of patients. Here, we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1. In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.

Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Ribosomal protein gene deletions detected by aCGH

Visual images of entire RP genes containing a deletion detected by αCGH analysis were made using Nimblegen’s SignalMap v1.9 software. A) RPL15 deletion in P1, B) RPS19 deletion in P2, C) RPS24 deletion in P3, D) RPS26 deletion in P4, E) RPS17 deletion in P5, F) RPS17 deletion in P6.

Fig. 2. RPS17 copy number measured by copy number assay

Copy number at exons 1, 3 and 5 of RPS17 was measured after normalization to RNase P in three control individuals, parents of P5, and in both DBA probands P5 and P6. Probands P5 and P6 showed ~50% reduced copy number at exons 1, 3 and 5 of RPS17 as compared to 3 control individuals and unaffected parents of P5.

Fig. 3. Analysis of pre-rRNA processing in cells derived from DBA patients

(A) Schematic representation of the human pre-rRNA primary transcript and its maturation intermediates. The endonucleolytic cleavage sites are indicated by arrowheads. The colored bars show the positions of the probes used in this work. The main precursors detected in the rest of the figure are schematized. (B) Polysome analysis on sucrose gradient shows decreased levels of free 60S subunits upon knockdown of RPL15, induced here with two different siRNAs. Half-mers are concomitantly detected in the polysomes (arrows), consistent with a defect in 60S subunit synthesis. Reduction of the RPL15 mRNA level was more efficient with the with the rpl15-2 siRNA (13% of the level observed with the scramble siRNA) than with the rpl15-3 siRNA (40%), as assessed by qRT-PCR, (C) Northern blot analysis of pre-rRNA processing in RPL15-depleted cells with the 5′ITS1, ITS2 and ITS1-721 probes. The ITS2 and ITS1-721 probes reveal the accumulation of 36S and 36S-C pre-rRNAs. The mature 18S and 28S rRNAs were detected with specific probes. (D) Pre-rRNA processing in LCL cells established from DBA patients was analyzed with the same probes as in C. A faint band corresponding to the 36S-C pre-rRNA is detected with the ITS1-721 probe in the RPL15 +/mut cells (P1), while 36S pre-rRNA is detected in the RPS17 +/mut sample (P5) (arrowheads). This is best seen on the intensity profiles of the upper part of the lanes shown on the left panel. The experiment was reproduced twice with similar results. (E) The Northern blot signals with the 5′ITS1 and ITS2 probes were measured by phosphorimaging, quantified using MultiGauge software and normalized to the 18S rRNA. For each species, the value of the mean of the two control samples was arbitrarily set to 1.