Ribosomal protein gene deletions in Diamond-Blackfan anemia

Abstract

Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

Figure 1

Single-copy deletions of RPS19 and RPS26 in DBA probands. Log2 intensity ratio of SNP probes surrounding deletions of RPS19 on chromosome 19 (A) and RPS26 on chromosome 12 (B). The regions displayed include chromosome 19 from 46 100 000-48 100 000 and chromosome 12 from 54 400 000-55 000 000, corresponding to the boxed region in the chromosome ideogram above each panel. The region of single-copy loss is shaded. The locations of RPS19 and RPS26 are indicated by the vertical line through the deletion regions.

Figure 2

Deletions involving RPS17 in DBA probands. (A) Log2 intensity ratio of SNP probes in the deletions adjacent to RPS17 on chromosome 15. The locations of 2 identical copies of RPS17 are indicated by the vertical lines. The identified region of copy loss is adjacent to RPS17; however, the paucity of probes, notably in the HumanOmniExpress chip (20QL and 1314), is apparent. (B) Log2 intensity ratios of CGH probes in the region of RPS17. Arrows at the top of the panel indicate the positions of RPS17. Gray points and connecting line depict a 30-kb median smoothing window. The region displayed is chromosome 15 from 79 450 000-83 800 000.

Figure 3

RPS17 copy number and expression by Q-RT-PCR. (A) Copy number at RPS17 was measured after internal normalization to an intergenic region of the β-hemoglobin locus in 3 normal control individuals, the 1314 and 20QL probands, and their parents. Normal copy number (ie, 4N) was defined as the average of 3 normal control individuals in each experiment. Both DBA probands showed reduced copy number at RPS17. (B) RPS17 expression levels were measured by internal normalization to RPL35A. Both deletion probands manifest haploinsufficiency of RPS17 mRNA, with levels consistent with approximately 50% expression. Graphs depict the average of 3 independent experiments, with each control and reference gene assayed in triplicate at each experiment. Normal expression (1.0) was taken as the average of 3 normal individuals in each experiment. Error bars depict the SEM. *P < .001 and **P < .0001 by unpaired t test compared with averaged normal controls.

Figure 4

Small subunit rRNA processing abnormalities in patients with RPS17 deletions. Northern blot analysis of rRNA with a hybridization probe to 18SE/ITS1 (probe γ) shows increased steady-state levels of 21S rRNA, indicating a functional ribosomal abnormality in patients with RPS17 deletions.

Figure 5

Variable mosaic copy loss in DBA gene regions. BAF plots of chromosome 3 (A) and 15 (B). The heterozygous region of the BAF is displayed, showing a normal individual with most heterozygous BAFs clustering about 0.5 (top panel). A progressively increasing mosaicism is evident in patient 802 (middle panel) as a splay in the heterozygous BAF region that increases approaching the telomere. Mosaicism is more obvious in 802-2 as a bifurcation of the heterozygous BAF region (bottom panel). (B) BAF plot from a normal individual's chromosome 15q (top panel). Chromosome 15 mosaicism is evident in patient 1786 (bottom panel). The regions displayed include the long arms of the respective chromosomes, corresponding to the boxed region in the chromosome ideogram. Dashed horizontal lines in the BAF plots indicated regions grouped for mosaicism analysis. The fraction of the population with monosomy in each analyzed regions is indicated above the BAF plot in each region analyzed and is outlined in the charts to the right. The illustration on the right depicts variable chromosome 15 mosaic monosomy corresponding to the fractional mosaicism identified for sample 1786.