A Large Ribosomal Subunit Protein Abnormality in Diamond-Blackfan Anemia (DBA).

DBA is an inherited bone marrow failure syndrome characterized by hypoproliferative anemia, congenital abnormalities and cancer predisposition. Ribosomal genes RPS19 and 24 are mutated in 25% and 3% of DBA patients respectively. To identify additional genetic abnormalities in DBA, we evaluated 2 unrelated children with DBA and sub-telomeric deletions of chromosome 3q by comparative genomic hybridization. The larger deletion spanned 11 Mb from 3q28 to the telomeric region and included 72 gene candidates. The second deletion involved 4 Mb from 3q29 to the telomeric end and included 52 known or hypothetical genes. The overlapping deletion region contained a previously reported 1.5 Mb microdeletion-associated syndrome that did not involve hematologic abnormalities, leaving 24 candidate genes. Gene expression microarray analysis from patient-derived EBV cell lines demonstrated down regulation of 7 of these candidate genes, one of which was RPL35a, a component of the large ribosomal subunit. We screened for mutations of RPL35a by direct sequencing of PCR-amplified genomic DNA from 149 DBA probands (125 sporadic, 24 familial) and 180 normal control subjects. We identified three probands with sequence changes in the RPL35a coding region: 1) an in-frame deletion in exon 3 (82-84CTT), causing a deletion of leucine at codon 28, 2) a nonsense mutation in exon 4 (298C>T), leading to an Arg102Stop and a 9 amino acid C-terminal truncation and 3) a missense mutation in exon 3 (97G>A) leading to a Val33Ile change. In the patient derived EBV cell line, the latter sequence change also resulted in an aberrant exon 3 splice site leading to a frame shift following codon 32. All of the probands with RPL35a mutations were sporadic cases. These sequence variations were not observed in the control subjects. Four lentiviral-based siRNA constructs targeting RPL35a were used to test the functional consequences of reduced RPL35a expression. Hematopoietic cell lines (TF-1 and UT-7/epo) transduced with the RPL35a directed siRNA constructs demonstrated decreased growth and viability compared to control siRNAs. Northern blot analysis demonstrated abnormal processing of large ribosomal subunit RNA with decreased mature 5.8S and 28S as well as decreased precursor 12S and 32S rRNA. Orthophosphate labeling confirmed a kinetic defect in large subunit rRNA processing, characterized by increased amounts of 45S and 41S rRNA with decreases of the precursors to and the mature 28S and 5.8S rRNAs. Mature 18S rRNA levels were unaffected, suggesting a defect in rRNA processing within the first internal transcribed sequence (ITS1). These data demonstrate that DBA can be caused by alterations in large as well as small ribosomal subunit proteins. These observations further support the hypothesis that altered ribosome homeostasis and function, rather than extra-ribosomal gene functions, is the central mechanism leading to DBA.