Exploiting pre-rRNA processing in Diamond Blackfan anemia gene discovery and diagnosis

Abstract

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene.

© 2014 Wiley Periodicals, Inc.

Figures

Figure 1

Photomicrographs of bone marrow aspiration films from the proband obtained at 6 months of age demonstrate marrow that is hypocellular for age (50-60% cellularity) with increased numbers of morphologically normal megakaryocytes, erythroid aplasia, myeloid hypoplasia with maturation arrest, eosinophilia and lymphocytosis (A, original magnification 200X and B, original magnification 600X). Plain film radiographs demonstrate proximal radioulnar synostosis (C) and right triphalangeal thumb (D). The 2q11.2 deletion identified in the proband is illustrated (E). The involved region, extending from 99,028,236 – 102,253,369 (hg19), is displayed relative to chromosome 2 and enlarged above, with annotated RefSeq genes indicated by white boxes and labeled by gene symbol. A large ribosomal subunit gene, RPL31 (black box, middle right), is included in the deleted region.

Figure 2

Northern blot analysis of pre-rRNA processing was performed on primary peripheral blood mononuclear cells from the proband, her parents, and a normal unaffected individual after culture for 96 hours in ConA (A and B) and on K562 erythroleukemia cells (C and D) after transduction with lentivirus expressing shRNA to RPL31 or luciferase (E). Membranes were interrogated with probes against ITS2 (A and C) and ITS1 (B and D), shown as * above schematic views of the pre-rRNA species to the right. In comparison to parents and a normal control, proband lymphocytes demonstrated a marked increase in 32S pre-rRNA abundance (A), indicative of a large subunit processing defect, as well as increased 45S and 18SE pre-rRNA, and diminished 30S pre-rRNA, suggesting involvement of small subunit processing (B). A similar pattern of abnormalities results from RPL31 knockdown in K562 cells (C and D), indicating that RPL31 haploinsufficiency alone is sufficient to produce these pre-rRNA processing abnormalities. Error bars indicate the SEM.

Figure 3

Elevated levels of 32S pre-rRNA, which are clearly evident by Northern analysis using a probe to ITS2 in RPL31-deleted proband RNA (A, upper panel), are also evident on visualization of the ethidium bromide-stained RNA gel (A, lower panel) from peripheral blood mononuclear cells after culture for 96 hours in ConA. Similar results were observed in DBA specimens harboring other large subunit alterations (B, demonstrating two independent RPL5 mutations) but not normal control RNA. Ethidium bromide-stained and Northern blot analysis of a larger sample of representative DBA patient RNA demonstrates a prominent 32S band in a patient with a known RPL11 mutation as well as a patient (DBA2) with an unknown gene mutation. 32S pre-rRNA is not elevated in control RNA or in a patient with mutation of the small ribosomal subunit gene RPS26. Another patient with unknown causative mutation (DBA1) lacks clear evidence for either large or small subunit processing defect. RNA was extracted from peripheral blood mononuclear cells after culture for 96 hours in ConA. Control lanes consist of RNA obtained from unaffected parents of the DBA proband (Panel A & B) or non-DBA healthy donors (Panel C).

Figure 4

Representative Agilent Bioanalyzer 2100 electrophoretogram data demonstrates prominent peaks corresponding to 18S and 28S rRNA, with a minor peak just larger than 28S rRNA corresponding to 32S pre-RNA, that was evident in analysis of total RNA from ConA-stimulated peripheral blood mononuclear cells (A). In comparison to non-DBA specimens (not shown) and DBA specimens with 40S gene alterations (A, upper panel), the 32S peak in DBA patients with 60S gene abnormalities is prominent (A, lower panel). The 32S/28S ratio in 7 tested DBA samples with 60S abnormalities is significantly higher than in 19 normal and 5 DBA specimens from patients with 40S subunit abnormalities (B). The two-tailed P-value of Mann Whitney, *** = 0.0001, ** = 0.0025, ns = 0.076.