De Novo Point Mutation of RPL31 in Diamond-Blackfan Anaemia: A Story from Patient to Zebrafish

Diamond Blackfan anemia (DBA) is a rare bone marrow failure disorder that usually manifests as severe macrocytic anemia and erythroid hypoplasia ,often accompanied by various physical malformations and increased frequency of cancer. The disease are associated with known ribosomal protein gene and rarely GATA1 mutations in most of DBA cases, with the genetic etiology unclear in the remaining patients.

Here, we present one patient with Diamond-Blackfan anaemia who lacked documented mutations involving known DBA genes by screening.Whole-exome sequencing (WES) was performed and we identified a single missense mutation in RPL31.The missense variant c.254G>T is predicted to give rise to a gene product with substitution p. Arg85Leu ,which is regarded as causative because the unaffected parents and sister of the patient do not possess the mutation,implying that this mutation was de novo. Previous studies showed that nearly all of the causative variants of RP genes observed in DBA are loss-of function mutations and RP haploinsufficiency accounts for most cases of DBA. Particularly, The coding region of RPL31 shares 89.6% amino acid identities with its zebrafish ortholog, which is highly conserved between these two species.All of above led us to explore the effects of RPL31 deficiency on morphology and erythropoietic status during embryonic development.

Therefore, morpholino antisense oligonucleotides (MOs) targeting zebrafish rpl31, orthologs of human RPL31,was injected at a proper concentration into one-cell-stage zebrafish embryos to mimic RPL31 haploinsufficiency in DBA. The specificity of the rpl31-MO was valitated by western blot detection.Compared with wild-type embryos, rpl31 deficiency embryos showed developmental abnormalities such as pericardial edema, microcephaly ,delayed pigmentation and bent tail. We also performed haemoglobin staining at 48 hr post fertilization (48hpf) and found a significant decrease of erythrocyte production in the cardial vein of the morphants. Meanwhile,the number of lcr+red blood cells was reduced about 50% in lcr:GFP transgenic embryos at 48 hpf by fluorescence microscopy and 3dpf by flow cytometry when compared to control embryos.However,the myeloid and lymphoid lineages were preserved as analyzed by using lyz:dsRed transgenic line and by observing expression of rag1,respectively. Lots of evidence showed that many other ribosomal protein genes deficiency can up-regulate p53 network,resulting in consequent cell cycle arrest or apoptosis.so we want to investigate the connection between RPL31 and p53 signaling in erythropoiesis.However,p53 null can not completely rescue the erythroid defect phenotype when we injected rpl31-MO into p53-/- embryos,suggesting alternative pathways may be involved in this process.

In summary, we reported a brand new point mutation in RPL31 gene and we phonecopied loss of function of RPL31 observed in the DBA patient in vivo by constructing a RPL31 deficiency zebrafish model,indicating preliminarily that the loss of function mutation in RPL31 is a candidate responsible for DBA.However,underlying molecular mechanisms and reasons of the specific erythroid impairments in RPL31 deficiency are still needed to be clarified.we will focused on these in the future.