RPS19 and JAK2 Are Not Silenced by DNA Methylation in Diamond-Blackfan Anemia.
Authors: Jelinek Jaroslav , Issa Jean-Pierre J. , He Rong , Cmejla Radek , Cmejlova Jana , Pospisilova Dagmar 
Published: April 2020

Abstract
Diamond Blackfan Anemia (DBA) is a congenital disorder characterized by decreased red blood cell production accompanied by developmental abnormalities in 30% patients. Twenty-five percent of DBA patients display heterozygous mutations of ribosomal protein S19 (RPS19) on chromosome 19q13.2. No mutations were found in genes for other ribosomal proteins of the translation initiation complex. Although a second DBA locus has been proposed on in the region 8p23.3–8p22, the precise molecular defect is not known in 75% of DBA patients. The exact mechanism of how RPS19 mutations affect erythropoiesis remains unclear. Haploinsufficency of RPS19 may hamper translation machinery important for rapid erythroid differentiation. Reduced gene expression of a cluster of ribosomal proteins including RPS19 in DBA patients was recently reported. No causal therapy for DBA is available, with the exception of bone marrow transplantation. Some DBA patients benefit therapeutically from corticosteroids, cyclosporine A, or metoclopramide. Recently, a long-lasting remission was described in a DBA patient treated with valproic acid, a histone deacetylase inhibitor, suggesting epigenetic suppression of genes critical for erythropoiesis may be involved in the pathogenesis of DBA. DNA methylation of promoter-associated CpG islands is an epigenetic modification resulting in transcriptional silencing functionally equivalent to a loss-of-function mutation. Constitutive activation of JAK2 tyrosine kinase by a somatic V617F mutation leads to excessive erythropoiesis in polycythemia vera, an antonym of DBA. We hypothesized that silencing by DNA methylation of promoter-associated CpG island of the RPS19 or JAK2 genes may play a role in DBA. To test the hypothesis, we analyzed DNA methylation of RPS19 and JAK2 genes in 14 patients from the Czech DBA Registry. Genomic DNA isolated from blood cells of 3 DBA patients carrying heterozygous RPS19 mutations, 11 DBA patients without RPS19 mutation and 4 control samples was treated with bisulfite to convert all unmethylated cytosines to uracils while methylated cytosines were spared from the conversion. A region spanning 13 CpG sites positioned from 1–160 bases downstream from transcription start site (TSS) of RPS19 gene was PCR amplified and cloned in a sequencing vector. Individual bacterial clones were isolated and PCR inserts were sequenced in 8–12 clones per sample. Bisulfite cloning and sequencing revealed that more than 99% of CpG sites were converted to TpG and thus not methylated either in DBA samples (only 4/1466 CpG sites were methylated, methylation frequency was 0.3%) or control samples (2/555 CpG sites methylated, methylation frequency 0.4%). To explore a possibility of epigenetic suppression of erythropoietin signaling in DBA we analyzed DNA methylation of the CpG island of JAK2 tyrosine kinase gene in the same set of samples. Bisulfite-treated DNA was PCR amplified and T/C polymorphisms corresponding to unmethylated or methylated CpG sites were quantified by pyrosequencing. All DBA and control samples showed the absence of DNA methylation at four CpG sites located 12 to 25 bases downstream of TSS. We conclude that epigenetic silencing by DNA methylation is not involved in the expression of ribosomal structural protein RPS19; neither it affects the expression of a transducer of erythropoietin signaling JAK2 tyrosine kinase.