Diamond-Blackfan Anemia (DBA) is characterized by a defect of erythroid progenitors and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis. By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes. Genotype–phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database.

Inherited and isolated nail malformations are rare and heterogeneous conditions. We identified two consanguineous pedigrees in which some family members were affected by isolated nail dysplasia that suggested an autosomal-recessive inheritance pattern and was characterized by claw-shaped nails, onychauxis, and onycholysis. Genome-wide SNP array analysis of affected individuals from both families showed an overlapping and homozygous region of 800 kb on the long arm of chromosome 8. The candidate region spans eight genes, and DNA sequence analysis revealed homozygous nonsense and missense mutations in FZD(6), the gene encoding Frizzled 6. FZD(6) belongs to a family of highly conserved membrane-bound WNT receptors involved in developmental processes and differentiation through several signaling pathways. We expressed the FZD(6) missense mutation and observed a quantitative shift in subcellular distribution from the plasma membrane to the lysosomes, where the receptor is inaccessible for signaling and presumably degraded. Analysis of human fibroblasts homozygous for the nonsense mutation showed an aberrant response to both WNT-3A and WNT-5A stimulation; this response was consistent with an effect on both canonical and noncanonical WNT-FZD signaling. A detailed analysis of the Fzd(6)(-/-) mice, previously shown to have an altered hair pattern, showed malformed claws predominantly of the hind limbs. Furthermore, a transient Fdz6 mRNA expression was observed in the epidermis of the digital tips at embryonic day 16.5 during early claw morphogenesis. Thus, our combined results show that FZD6 mutations can result in severe defects in nail and claw formation through reduced or abolished membranous FZD(6) levels and several nonfunctional WNT-FZD pathways.

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a specific decrease of erythroid precursors. The disease is also associated with growth retardation, congenital malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. We show herein that primary fibroblast from DBA patients with truncating mutations in RPS19 or in RPS24 have a marked reduction in proliferative capacity. Mutant fibroblasts are associated with extended cell cycles and normal levels of p53 when compared to w.t. cells. RPS19 mutant fibroblasts accumulate in the G1 phase, whereas the RPS24 mutant cells show an altered progression in the S phase resulting in reduced levels in the G2/M phase. RPS19 deficient cells exhibit reduced levels of Cyclin-E, CDK2 and retinoblastoma (Rb) protein supporting a cell cycle arrest in the G1 phase. In contrast, RPS24 deficient cells show increased levels of the cell cycle inhibitor p21 and a seemingly opposing increase in Cyclin-E, CDK4 and CDK6. In combination, our results show that RPS19 and RPS24 insufficient fibroblasts have an impaired growth caused by distinct blockages in the cell cycle. We suggest this proliferative constraint to be an important contributing mechanism for the complex extra-hematological features observed in DBA.

a b s t r a c tRibosomal protein S19 (RPS19) is mutated in patients with Diamond-Blackfan anemia (DBA). We hypothesized that decreased levels of RPS19 lead to a coordinated down-regulation of other ribosomal (r-)proteins at the subunit level. We show that small interfering RNA (siRNA) knock-down of RPS19 results in a relative decrease of small subunit (SSU) r-proteins (S20, S21 and S24) when compared to large subunit (LSU) r-proteins (L3, L9, L30 and L38). This correlates with a relative decrease in 18S rRNA with respect to 28S rRNA. The r-protein mRNA levels remain relatively unchanged indicating a post transcriptional regulation of r-proteins at the level of subunit formation.