The First Homozygous RPS19 Mutation Identified in a Diamond-Blackfan Anemia Patient Is Not Deleterious.

Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA). Two of the RPS19 mutations identified in DBA patients are located in the sequence upstream of the translational start site: a missense mutation g->t at −460 and a 4 bp insertion, gcca, 3 nt upstream of the 5′ end of the RPS19 promoter. All of the RPS19 mutations identified to date are expressed in the heterozygous state. In the present study, in a DBA patient from Ivory Coast we identified the gcca insertional mutation on both alleles. Diagnosis of DBA was made at the age of 3 months. No malformations were noted. The patient responded neither to steroid nor to IL-3 therapies. The patient was regularly transfused but without an effective iron chelation therapy. As a consequence DBA was complicated by a severe hemochromatosis with thyroid, parathyroid, and liver damage. The patient was enrolled into a metoclopramide protocol in our hospital when he was 12 year old. He had a partial response, with increased time intervals between transfusions. While analysis of the RPS19 gene did not identify a mutation in the coding sequence, the 4 bp insertion, gcca at nt − 631 was noted on both alleles. Mother was heterozygous for this mutation and strikingly the father was homozygous. Both parents are apparently healthy. In screening of 200 Caucasian control chromosomes and 100 chromosomes from Ivory Coast we did not identify the 4bp insertion, ruling out the possibility that it is a common polymorphism. A parental disomy was eliminated by a genescan microsatellite analysis using the microsatellites: D19S200, D19S197, and LIPE. To evaluate the effect of this mutation on erythroid differentiation, we isolated 30,000 CD34⁺ cells from peripheral blood of the patient and normal individuals. CD34⁺ cells were cultured for 7 days in methylcellulose with EPO, SCF, and IL-3. At day 7, erythroid colonies were isolated and cultured for additional 3 and 5 days in liquid medium. At D7, D10 and D12, aliquots of cells were collected and cloning efficiency and apoptosis was quantitated. RPS19 mRNA was assayed by quantitative RT-PCR and level of protein expression determined by Western blot analysis. The cloning efficiency of DBA erythroid progenitors was decreased by 2.6 fold compared to normal between D7 and D10 and by 3.5 fold between D10 and D12. No difference in apoptosis was noted between DBA and normal erythroid progenitors. Strikingly, during terminal erythroid differentiation, RPS19 mRNA and protein expression levels was similar in the DBA and normal erythroid cells. The presence of the homozygous mutation in the healthy father in conjunction with normal expression of RPS19 in the DBA erythroid cells imply that the gcca insertion at nt −631 is not deleterious and cannot by itself account for the DBA phenotype of our patient.