Diamond-Blackfan anemia: genotype-phenotype correlations in Italian patients with RPL5 and RPL11 mutations

Abstract

Background: Diamond-Blackfan anemia is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of patients display various malformations. Anemia is corrected by steroid treatment in more than 50% of cases; non-responders need chronic transfusions or stem cell transplantation. Defects in the RPS19 gene, encoding the ribosomal protein S19, are the main known cause of Diamond-Blackfan anemia and account for more than 25% of cases. Mutations in RPS24, RPS17, and RPL35A described in a minority of patients show that Diamond-Blackfan anemia is a disorder of ribosome biogenesis. Two new genes (RPL5, RPL11), encoding for ribosomal proteins of the large subunit, have been reported to be involved in a considerable percentage of patients.

Design and methods: In this genotype-phenotype analysis we screened the coding sequence and intron-exon boundaries of RPS14, RPS16, RPS24, RPL5, RPL11, and RPL35A in 92 Italian patients with Diamond-Blackfan anemia who were negative for RPS19 mutations.

Results: About 20% of the patients screened had mutations in RPL5 or RPL11, and only 1.6% in RPS24. All but three mutations that we report here are new mutations. No mutations were found in RPS14, RPS16, or RPL35A. Remarkably, we observed a higher percentage of somatic malformations in patients with RPL5 and RPL11 mutations. A close association was evident between RPL5 mutations and craniofacial malformations, and between hand malformations and RPL11 mutations.

Conclusions: Mutations in four ribosomal proteins account for around 50% of all cases of Diamond-Blackfan anemia in Italian patients. Genotype-phenotype data suggest that mutation screening should begin with RPL5 and RPL11 in patients with Diamond-Blackfan anemia with malformations.

Figure 1.

Pedigree of a DBA patient carrying mutations in both the RPL5 and RPS24 genes.

Figure 2.

Transient transfection of RPS24 constructs. (A) Total extracts from HEK293 cells transfected with plasmids coding for RPS24 WT (WT) and mutated forms of the protein (M1, M2) were separated by SDS-PAGE and transferred on nitrocellulose membrane. The blots were decorated with anti-Flag (Flag) and anti-neomycin phosphotransferase II (NPT) antibodies. Quantitation of the signals is reported in the lower part as the ratio of Flag/NPT; (B) Total extracts from transfected cells (as in A) were separated into nuclear (N) and cytoplasmic (C) fractions. The loading ratio between cytoplasmic and nuclear extracts was 1:20 (number of cells). Western blotting was performed as in A with anti-Flag (Flag) and anti-RPS19 (RPS19). (C) Cytoplasmic extracts from HEK293 cells transfected as in A were fractionated by ultracentrifugation on a sucrose cushion as indicated in the Design and Methods section. Pellets, which include ribosome and ribosomal subunits (P) and trichloroacetate-precipitated supernatants, which include free cytoplasmic proteins (S) were analyzed by western blot with anti-Flag (Flag) and anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibodies.

Figure 3.

Malformation status of patients with RPL5 and RPL11 mutations. Associations between malformation status and RP gene mutations were assessed with odds ratio (OR) and 95% CI calculated from logistic regression; OR are drawn on a logarithmic scale. RPL5 and RPL11-mutated patients were compared to both RPS19-mutated patients and non-mutated patients. The Wald test was used to test the statistical significance of each association, the P value is indicated as (*) P<0.05; (§) P<0.01.