Molecular pathology of Diamond Blackfan anaemia

Diamond-Blackfan anaemia (DBA) is one of a rare group of genetic disorders known as ‘inherited bone marrow failure (IBMF) syndromes with bone marrow failure, birth defects and higher propensity to cancer. It is arare autosomal dominant disorder with an incidence of 7 in 106 newborns, DBA is characterized by a defect in erythroid lineage development and a quantitative as well as a qualitative defect in erythroid progenitors. This disorder is inherited in 45% of cases and emerges as de novo in the remaining cases. Diagnosis of DBA is challenging as it is based on several clinical features shared by other IBMF syndromes. Diagnosis of this disorder has remained for many years a challenge. In recent years, the use of Next generation sequencing (NGS) of the 83 ribosomal protein (RP) genes has greatly improved the diagnosis and the future prospect of managing the disease. This new diagnostic approach provides several advantages over other conventional and classic approaches, last but not least that now over 65-80% of DBA cases have been identified to carry a genetic mutation of one of the 83 RP genes, while only few could be analyzed until recently. It also facilitates the analysis of DBA family members to facilitate both the selection of possible donors for transplant and to distinguish inherited from de novo mutations. RP gene mutations leading to reduced amounts of RPs (affecting both the 40S and 60S ribosomal subunits) interfere with the processing of rRNA. Until recently the different rRNA species in DBA patients could only be analyzed using Northern blot technique, with several limitations mainly due to the poor yield of RNA in these patients. To resolve some of these problems, in this study we designed primers specific for 32s rRNA intermediate, 18s, 28s, and 5s rRNA. We studied the relative expression of these genes in DBA compared to healthy controls. We studied rRNA defect in a cohort of forty-eight DBA patients from resting and stimulated T cells. rRNA profile study showed a significant difference in the resting and stimulated T cells from DBA patients compared to controls. We then applied CRISPR CAS 9 technology, electroporation, flowcytometry, and timed cell-sorting to validate two novel heterozygous mutations discovered in our Lab, the RPS17 c.3G>C and RPL11 c.475_476 del AA. We optimized a method to successfully introduce a heterozygous knock in mutation using CRISPR/Cas9 technology in K562 erythroleukemic cell line. Firstly, K562 cells were transfected with LentiCRISPRv2 plasmid that has a specific guide RNA (gRNA) ligated to it using the Amaxa nucleofection system. Seventy-two hours later, single green fluorescent protein (GFP) expressing K562 cells were sorted into 96 wells plates. After three weeks of growing these single clones, they were analyzed and the viability was shown to be 50%. Sanger sequencing was carried out to confirm the presence of heterozygous knock in of the specific mutation. Quantitative real time PCR studies using our designed primers (18S, 28S, 32S and 5.8S rRNA) revealed that both mutations resulted in rRNA processing defect in K562 cell line compared to control. In conclusion, our study lead to the characterization of newly established mutations and we demonstrated that such mutations were responsible and causative of a defect in the production of rRNA in an in vitro model.Open Acces