Expansion of germline RPS20 mutation phenotype to include Diamond-Blackfan anemia

Abstract

Diamond-Blackfan anemia (DBA) is a ribosomopathy of variable expressivity and penetrance characterized by red cell aplasia, congenital anomalies, and predisposition to certain cancers, including early-onset colorectal cancer (CRC). DBA is primarily caused by a dominant mutation of a ribosomal protein (RP) gene, although approximately 20% of patients remain genetically uncharacterized despite exome sequencing and copy number analysis. Although somatic loss-of-function mutations in RP genes have been reported in sporadic cancers, with the exceptions of 5q-myelodysplastic syndrome (RPS14) and microsatellite unstable CRC (RPL22), these cancers are not enriched in DBA. Conversely, pathogenic variants in RPS20 were previously implicated in familial CRC; however, none of the reported individuals had classical DBA features. We describe two unrelated children with DBA lacking variants in known DBA genes who were found by exome sequencing to have de novo novel missense variants in RPS20. The variants affect the same amino acid but result in different substitutions and reduce the RPS20 protein level. Yeast models with mutation of the cognate residue resulted in defects in growth, ribosome biogenesis, and polysome formation. These findings expand the phenotypic spectrum of RPS20 mutation beyond familial CRC to include DBA, which itself is associated with increased risk of CRC.

Trial registration: ClinicalTrials.gov NCT00027274.

Keywords: Diamond-Blackfan anemia; RPS20; familial colorectal cancer; ribosomopathy; yeast model.

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

The authors report no competing interests.

© 2020 Wiley Periodicals LLC.

Figures

Figure 1

Previously reported and two novel RPS20 germline variants. (A) Positions of germline RPS20 variants previously reported in familial colorectal cancer (black), a young adult with metachronous CRC (blue), and a young adult predicted to have Lynch syndrome (green) (Broderick et al., 2017; Nieminen et al., 2014), as well as those identified in BMF92 and NCI-62–1 (red). (B) BMF92 and NCI-62–1 family trees and sequence chromatograms of RPS20 genomic DNA from buccal swab (BMF92) or LCL (NCI-62–1). A half-filled symbol indicates the presence of one variant.

Figure 2

The structure of RPS20 in the human 80S ribosome and reduced expression of mutant RPS20 proteins (A) The localization of RPS20 in the human 80S ribosome (PDB ID: 6EK0) (Garreau de Loubresse et al., 2014; Natchiar et al., 2017). The 80S ribosome and RPS20 are colored cyan and yellow, respectively. (B) Close-up view of RPS20 p.Ile84 in the human 80S ribosome. 18S ribosomal RNA, RPS3, and RPS29 are colored orange, wheat and green, respectively. The amino acid residues located around p.Ile84 are depicted by stick models, and the corresponding amino acid residues are labeled. (C) Graphical representation of the conserved motif within which human p.Ile84 resides [generated by WebLogo (Crooks, Hon, Chandonia, & Brenner, 2004)]; p.Ile84 is indicated by the arrowhead. (D) Representative western blot of RPS20 protein in LCL whole cell lysates from proband BMF92 compared to his unaffected father, BMF92-F, or sibling BMF92-S1. β-actin is a loading control. (E) Representative western blot of wild type, p.Ile84Asn-, or p.Ile84Ser-mutant RPS20 protein in 293T whole cell lysates following transient transfection with the designated quantity of myc-RPS20 plasmid. Neomycin and β-actin are transfection and loading controls, respectively.

Figure 3

RPS20 p.Ile84Asn missense variant LCL has altered ribosome and polysome profiles. Polysome profile analysis of (A)RPS20 unaffected sibling control (BMF92-S1), BMF92 (RPS20 mutated), and RPS19 mutant (BMF51). Close-up views of (B) BMF92-S1, (C) BMF92, and (D) BMF51 LCL polysome profile analyses. (E) Composite of graphs B-D. Positions of the 40S, 60S, 80S and polysome peaks are shown.

Figure 4

Mutation of yeast Rps20 p.Ile86 can affect growth, ribosome biogenesis, and polysome profiles. (A) Superposition of human RPS20 (yellow) and S. cerevisiae Rps20 (light blue) structures. Human Ile84/S. cerevisiae Ile86 are depicted by stick models. (B) Close-up view of Rps20 p.Ile86 interactions in the S. cerevisiae (yeast) 80S ribosome (PDB ID: 4U4R) (Garreau de Loubresse et al., 2014; Natchiar et al., 2017); 18S ribosomal RNA, Rps3, Rps29 and Rps20 are colored light yellow, pink, magenta, and light blue, respectively. (C) S. cerevisiae expressing wild type and mutant Rps20 proteins plated at 1:10 serial dilutions and grown at the designated temperatures for the designated times. (D) Polysome profiling of yeast bearing RPS20 (wild type), rps20-Ile86Asn, or rps20-Ile86Asp alleles. Area under the curve 60S/40S and polysome/80S ratios are shown. The rps20-Ile86Asn strain was analyzed using one transformant, whereas the wild type and rps20-Ile86Asp strains were analyzed using two transformants, with the averages and standard deviations shown.

Figure 5

RPS20 p.Ile84 missense variant LCLs have variably altered pre-rRNA processing. (A) Representative electropherograms from LCL total RNA. The 18S and 28S rRNA peaks are indicated, as well as a minor 32S pre-rRNA peak in the BMF16 sample. (B) Comparison of 28S/18S rRNA ratios as measured by electropherograms; similar symbols came from the same experiment. P values were not calculated for BMF92-S1 nor NCI-62–1 as fewer than three biological replicates were performed. Shown is the mean and standard error of the mean (sem). (C) Representative northern blot analysis of RNA isolated from the designated LCLs (listed below) using an ITS1 probe. (D) Quantification of 21S pre-rRNA/28S rRNA ratios from four independent blots as in C. (E) Quantification of 18S-E pre-rRNA/28S rRNA ratios from four independent blots as in C. (F) Quantification of 21S/18S-E pre-rRNA ratios from four independent blots as in C. LCLs tested: BMF92 and NCI-62–1: RPS20 missense variants; BMF16: RPL5 missense variant; BMF92-F: unaffected father of BMF92; BMF92-S1: unaffected sibling of BMF92; RQ4–115: healthy/unaffected control; BMF54 and BMF73: RPS19 missense variants.