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RPL15 mutations in DBA
mutant cells as compared to healthy controls (Figures 5C and Online Supplementary Figure S5). In addition, Tyr81* mutant cells retain a higher expression of the IL3 receptor that is normally down-regulated in the erythroid lineage, and express less erythroid-specific glycophorin A (GPA), Band-3, or alpha-4 integrin compared to healthy control cells (Figure 5D and E and Online Supplementary Figure S5). Additionally, expression of RPL15 mRNA was lower (Figure 5F) and of p21 mRNA higher in Tyr81* mutant cells (Figure 5G). The observed p21 overexpression is like- ly TP53-mediated, as supported by western blot analysis confirming an increase in TP53 phosphorylation and increased p21 protein expression (Figure 5H). Finally, TP53 protein stabilization was observed in Tyr81* mutant patient cells (Figure 5I).
Discussion
At present there are 19 RP genes associated with DBA, representing almost one-quarter of the 80 cytoplasmic RPs in human cells. Here, we describe a novel genetic subgroup of DBA due to mutations in the RPL15 gene and identify the truncating mutation c.242dupA; p.Tyr81* as a recurrent genetic cause in 3 unrelated patients. The experimental results show that LCLs carry- ing the p.Tyr81* variant reveal an array of molecular defects typically associated with a ribosomopathy phe- notype. These include impaired pre-rRNA processing, reduced 60S ribosomal subunit formation, a reduction in de novo protein synthesis, and impaired cell proliferation. Further, mutant LCLs phenocopied biological changes observed in HeLa cells depleted of eL15. These results, similar to previous reports that DBA-linked RP muta- tions impair pre-rRNA processing, indicate that the RPL15 mutations reported here are likely pathogenic and result in haploinsufficiency.
The altered ribosome biogenesis may explain the observed reduction in global protein synthesis, substanti- ating previous reports discussing altered translation of spe- cific mRNAs in DBA-mutant cells as a key component of disease pathogenesis.49,50 In support of this pathogenesis, we also found that hematopoietic stem cells with the eL15 Tyr81* variant that were induced to differentiate into ery- throcytes revealed a substantial decrease in the number of erythroid colonies and delays in differentiation. These findings, in addition to increased apoptosis, TP53 stabi- lization, and p21 overexpression in RPL15-mutant hematopoiesis, suggest that eL15 plays a critical role in ribosome biogenesis and that the reduction of the protein by genetic haploinsufficiency drives severe stress in the nucleolus.
Hydrops fetalis arising from severe intrauterine anemia is an uncommon manifestation of DBA with 10 cases reported so far. The clinical outcome of these patients was poor and, unexpectedly, no spontaneous remission was observed. In contrast, 50% (3 of 6) of all patients with RPL15 mutations reported in our study showed prenatal manifestation with the necessity of intrauterine transfu- sions. Remarkably, the hydrops cases were observed only in the subgroup of patients carrying protein truncating mutations p.Tyr81* and p.Gln29*, with prevalence of hydrops reaching 75% (3 of 4).
A substantial percentage of individuals with DBA spon- taneously achieve treatment independence at some point; however, the reasons for this remain elusive and no pre- dictive biomarkers exist.39 In line with this, another unex- pected finding was the rapid and sustained treatment independence achieved within four to 16 months after birth in all 3 unrelated DBA patients with the recurrent p.Tyr81* mutation. Although Patient 1 relapsed five years after achieving spontaneous remission, a short steroid course was very effective and resulted in treatment inde- pendence. These observations suggest that p.Tyr81* mutation carriers can achieve treatment independence with or without a previous course of steroids, and that a genotype itself may be an important biomarker for the expected clinical course. Our findings might help prospec- tive clinical stratification to determine which individuals are more likely to become treatment independent without the need for HSCT. The reasons why the specific muta- tion p.Tyr81* drives treatment independence remain unknown and cannot be explained by a potential genetic reversion, which was not observed in our patients. One tempting speculation is that the remaining wild-type allele compensates for the haploinsufficiency (e.g. by epigenetic mechanisms) in the adult hematopoiesis, but the compen- sation might not be present or sufficient in the fetus. These findings warrant further studies on the future avail- ability of more patient material.
In conclusion, our study establishes germline point mutations in the RPL15 gene as novel genetic etiology of DBA. Half of the individuals carrying these mutations manifest with hydrops fetalis, a phenotype that is very rarely observed in DBA patients. Finally, we establish that a recurrent DBA genotype, eL15 p.Tyr81*, is linked to treatment independence.
Acknowledgments
Very special thanks go to all the individuals who consented to participate in this study and their families. We thank Alexandra Fischer (Freiburg) for data management, Sandra Zolles, Sophia Hollander, Dirk Lebrecht, and Gunda Ruzaike (Freiburg) for technical assistance, Pritam Kumar Panda (Freiburg) for bioinfor- matic analysis, and Dr. Marije Bartels (UMC Utrecht) for criti- cal reading of the manuscript.
Funding
AM, ML, MW, LDC, NK, HT, PEG, and MFOD are sup- ported under the framework of E-Rare-2, ERA-Net for Research on Rare Diseases (ZonMW #113301205 and #40-44000-98- 1008 in the Netherlands; #BMBF 01GM1301 and 01GM1609 in Germany; Chief Scientist Office, Israeli Ministry of Health # 3-12844 in Israel; #ANR-15-RAR3-0007-04 and #ANR-12-RARE-0007-02 in France). MW and CN are sup- ported by DKTK German Cancer Consortium, fot molecular diagnostics of pediatric malignancies. LDC was supported by the Laboratory of Excellence for Red Cells [(LABEX GR-Ex)-ANR Avenir-11-LABX-0005-02], the French National PHRC OFABD (DBA registry and molecular biology), and the “Fondation ARC pour la recherche contre le cancer”. LDC, MFOD and PEG are funded by the French National Research Agency [ANR-DBA-Multigenes-ANR-2015-AAP générique- CE12]. AA was supported by DBA Telethon grant GGP13177 and Fondazione Europea per la DBA and Gruppo di Sostegno DBA Italia.
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