Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia

Abstract

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in approximately 30%-50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.

Figure 1

RNA Blot Analysis of the Effects of RPS10 and RPS26 Mutation on Pre-rRNA Processing (A) HeLa cells were transfected with siRNAs to knock down RPS10 and RPS26 expression. RNAs extracted 48 hr posttransfection were probed with oligonucleotides complementary to the 5′ end of the ITS1 (5′ ITS1), which detects the precursors to the 18S rRNA or to the mature 18S and 28S rRNAs. Transfected cells show similar phenotypes with high levels of 43S, 26S, and 18S-E RNA, together with low levels of 18S rRNA. Similar results were obtained with two independent siRNAs for each gene. (B) Total RNAs from lymphoblastoid cells derived from DBA patients (Pt) and unaffected relatives were analyzed as in (A). Controls (Ct) correspond to cells from unaffected individuals unrelated to patients. RNAs from patients mutated in RPS19 are also shown for comparison. The last lane on the right corresponds to an individual bearing a missense change in RPS10, most likely a rare normal variant. (C) Phosphorimager analysis of the 18S-E and 21S pre-rRNA levels in RPS26^+/mut patients shows that four out of five patients have elevated levels of 18S-E rRNA when compared to their unaffected relatives. In each lane, measurements were normalized to the amount of 28S rRNA and divided by the mean of the corresponding value in the three controls. (D) Relative abundance of the 18S RNA precursors was quantified by phosphorimaging in DBA patients mutated in RPS10, RPS26, and RPS19. Controls in this panel correspond to all the unaffected individuals shown in (B), both patient relatives and unrelated persons.