Two Novel Mutations in Ribosomal Proteins in the Czech National Diamond-Blackfan Anemia Registry
Authors: Maceckova Zuzana , Koralkova Pavla , Volejnikova Jana , Vojta Petr , Jungova Alexandra , Saxova Zuzana , Mojzikova Renata , Jarosova Marie , Hadacova Ivana , Hajduch Marian , Horvathova Monika , Pospisilova Dagmar 
Published: October 2019

Abstract
The Czech National Diamond-Blackfan Anemia (DBA) Registry currently consists of 51 patients; in 36 of them ribosomal protein (RP) mutations have previously been identified. Targeted sequencing of genes encoding RP, exomes and transcriptomic sequencing and/or array CGH were performed in newly diagnosed DBA patients and in patients without a causative mutation. Two novel mutations in RPS7 and RPL11 were discovered in 5 individuals from two families.
Aheterozygous mutation g[3580153G>T] in exon 6 of a gene coding for RPS7, leading to V134F substitution, was found in an 18-year-old female who was transfusion dependent during infancy, responded to steroids and is currently in remission. The identical mutation was detected in the patient's mother and older sister, who both are asymptomatic with borderline hemoglobin (Hb) levels, mild macrocytosis with no active treatment. A functional study using cellular models revealed that RPS7-deficient MRC-5 fibroblasts transiently expressing RPS7-V134F exhibit altered protein synthesis, defective ribosomal RNA processing, increased nucleolar and ribosomal stress. This was evidenced by overexpression and translocation of p53 protein to the nucleus in RPS7-V134F but not RPS7 wild type transfected cells. These data are consistent with previously reported cellular phenotypes in DBA. In addition, increased levels of erythrocyte adenosine deaminase (e-ADA) were detected in all three family members with V134F RPS7 mutation (patient: 4.2±0.8 IU/g Hb; mother: 3.5±0.2 IU/g Hb; sister: 4.6±0.3 IU/g Hb; reference range: 0.8-2.5 IU/g Hb). Recently it was shown that ribosomal insufficiency increases oxidative stress in red blood cells (RBC) of DBA patients. In this family, increased levels of reactive oxygen species (ROS) were detected by flow cytometry only in patient's RBC, but not in RBC of her asymptomatic mother and sister. Consistently, only the patient showed increased anti-oxidative defense parameters compared to the controls and her mother and sister. Markedly elevated levels of reduced glutathione (GSH) and glucose 6-phosphate dehydrogenase (G6PD), two essential antioxidants, were detected in patient's RBC (GSH: 4356±44 mM; G6PD: 10.3±0.6 IU/g Hb) compared to controls (GSH: 2434±454 mM; G6PD: 5.4-7.0 IU/g Hb) and patient's mother (GSH: 2125 mM; G6PD: 5.3±0.2 IU/g Hb) and sister (GSH: 2450±35 mM; G6PD: 6.5±0.1 IU/g Hb). Concomitant increase (approximately 2.5 times) in the activity of hexokinase (HK) and pyruvate kinase (PK) and in the levels of ATP in patient's RBC indicates augmented energy metabolism to sustain the integrity of RBC. Flow cytometry of Annexin V binding revealed increased exposure of phosphatidylserines on RBC membrane of the patient compared to controls and her mother and sister suggesting that the tendency towards redox balance restoration and membrane integrity does not completely prevent an enhanced recognition and destruction of patient's RBC by reticuloendothelial macrophages.
The second c.281T>G point mutation in RPL11 was found in a 34-year-old woman with unspecified macrocytic anemia, who developed diffuse large B-cell lymphoma (DLBCL). She was transfusion dependent in infancy, responded to steroids and is currently in the anemia remission and second remission of DLBCL. Identical mutation was detected in her 56-year-old mother, followed for macrocytic anemia and gammopathy.
In 10 DBA cases without the causative mutation an array CGH was performed, but no large deletions in RPS19, RPS17, RPS26, RPL5 and RPL11 genes were detected.
In summary, we present the first case of a missense mutation in RPS7 as the cause of DBA; all to date published RPS7 mutations clinically associated with DBA were located in splice sites. Similarly, the co-occurrence of DBA and DLBCL has not been previously published. We also emphasize that the phenotype of family members with the same mutations could be different, including silent carriers. All family members should therefore be examined even if asymptomatic. Testing of eADA levels could serve as a sensitive screening method in apparently unaffected family members. Finally we suppose that altered RBC metabolism may affect the lifespan of RBC in DBA and thus contribute to the worsening of the blood condition, especially during infections. The analyses in a large cohort of DBA patients are ongoing.
Grant Support: AZV16-32105A, GA15-13732S, MH CZ-DRO FNOL 00098892, NPU LO 1304, IGA UP LF_2016_014.

Disclosures
No relevant conflicts of interest to declare.