Abstract

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (BMFS) affecting one in 105 live births. In the majority of patients the disease results from heterozygous mutations or deletions in genes encoding ribosomal proteins (RP), suggesting haploinsufficiency for RPs causes the disease. DBA presentation and outcome is very variable, even in the same family, with 20% of patients going into remission by an unknown mechanism. In some cases remission in BMFS has been associated with clonal expansion of cells bearing acquired genomic changes. We hypothesized that a similar mechanism may underlie remissions in DBA. In order to detect somatic single nucleotide mutations, copy number variations (CNV), and copy neutral loss of heterozygosity (CN-LOH) in the bone marrow of DBA patients, we took advantage of whole exome sequencing (WES) and single nucleotide polymorphism (SNP) array technologies. Genotyping was performed on paired DNA samples from bone marrow and skin fibroblasts of five independent patients with DBA. About 30 non-synonymous coding differences per sample pair (skin and BM) were called. These were reduced by eliminating 1. mutations due to technical and bioinformatics artifacts that often appear in these data. 2. mutations that were not validated by Sanger sequencing. While analysis and validation is ongoing we have positively identified 2 candidate gene mutations that were found in DNA from BM cells but not from skin fibroblasts from 2 individuals.

In the case discussed here a somatic mutation in USP11, a c.G1253A leading to a R418Q amino acid substitution, was detected by WES in a DBA male patient with a relatively mild disease. Sanger sequencing validated this finding in the bone marrow and the peripheral blood of the patient. USP11 is located on the X chromosome and the R418Q substitution is predicted to profoundly affect the catalytic subunit of USP11, according to in-silico analysis.

We hypothesized that the USP11 mutation may have favorably affected the patient's hematopoiesis, leading to a milder form of DBA, due to a compensatory effect on ribosomal biogenesis. To determine the effects of this mutation on RPs we took advantage of lymphoblastoid cell lines (LCLs) from DBA patients. The LCLs of two DBA patients with heterozygous RPL5 mutations were examined along with control LCLs. One of the DBA LCL lines was hemizygous for USP11R418Q and the other was WT at the USP11 locus. The levels of RPL11 and RPL5 were higher in cell lines from the patient hemizygous for USP11R418Q when compared with those from the patient with WT-USP11. Furthermore, levels of RPL5 and RPL11 in the patient carrying the USP11R418Q allele were similar to those of a healthy control. RPL11 is important in regulating the MDM2/p53 pathway implicated in DBA pathogenesis. In a second set of experiments we overexpressed mutant and wild type USP11 in HEK293T cells and observed that ribosomal protein RPL11 was again overexpressed compared with when WT USP11 was mutated. Overexpression of mutant USP11 led to the accumulation of ubiquitinated proteins, suggesting the mutation interfered with deubiquitination.

USP11 is known to play a role in DNA repair, the TNF-α and the TGFβ pathways. The precise mechanism of action of USP11 in controlling the level of RPL11 is still obscure. Nevertheless, our findings suggest that USP11 may play an important role in the regulation of RP levels and may be a candidate for investigation as a therapeutic target in DBA.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.