Identification of New Rare Sequence Changes in RP Genes in Diamond-Blackfan Anemia and Association of the RPL5 and RPL11 Mutations with Craniofacial and Thumb Malformations

Diamond-Blackfan anemia (DBA), a congenital bone marrow failure syndrome, is characterized by red blood cell aplasia, macrocytic anemia, clinical heterogeneity, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies, present in ~30–47% of patients. The disease is associated with mutations in six ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, and L11, in about 40–45% of patients. To continue our large scale screen of RP genes in a DBA population, we sequenced 12 RP genes, S15, L36, L31, L37A, S7, S27A, S14, S23, L3, L23, S17, and L27A in our DBA patient cohort of 200 families. We identified the second known mutation in RPS17 and possible pathogenic single mutations in four more RP genes, S7, L36, S15, and S27A. These are a donor splice site mutation (intron 2) in RPS7, a deletion of two nucleotides causing frameshift in RPS17 and RPL36, and two missense changes in RPS15 and RPS27A. Northern blot analysis demonstrated that lymphoblastoid cells from the patient with RPS7 mutation displayed higher levels of 45S and 30S pre-rRNAs compared to normal cells, similar to results in HeLa cells with siRNA-based knock-down of RPS7. There is a strong defect in 5′-ETS processing, resulting in accumulation of 45S and 30S pre-rRNAs, and a strong drop of levels of the 41S, 21S and 18S-E intermediates, whereas the amount of precursors to the large ribosomal subunit RNAs were unchanged. These results suggest that mutation of RPS7 in this DBA patient directly affects maturation of pre-rRNA. In addition, review of available medical data of 20 patients with mutations in RPL5 revealed that majority of them (14/20) have physical malformations including craniofacial, thumb and heart anomalies. Similarly, 12/18 patients with RPL11 mutations presented with physical malformations, while among 76 reported DBA patients with RPS19 mutations, only 35 (46%) had physical abnormalities. Remarkably, 9 of 14 patients with RPL5 mutations and physical abnormalities have cleft lip/ palate or cleft soft palate, isolated or in combination with other facial malformations and/or with other physical abnormalities such as heart or thumb anomalies. In contrast, none of 12 patients with RPL11 mutations and malformations have craniofacial abnormalities (p=0.007, Fisher’s exact test [FET]). Moreover, none of the 35 reported patients with RPS19 mutations and malformations presented with cleft lip and/or palate (p=9.745×10⁻⁷for RPL5 vs RPS19, FET). We conclude craniofacial clefting is associated with mutations in RPL5. In addition, 8/20 patients with mutated RPL5 and 8/18 patients with mutated RPL11 have thumb abnormalities, compared to only 9% of patients with RPS19 mutations. Moreover, congenital heart defects were found more often among patients with RPL5 mutations (5/20) compared with RPL11 (3/18) and RPS19 (4/76) (p=0.017 for RPL5 vs RPS19, FET). Strikingly, the majority (11/20) of patients with RPL5 mutations presented with multiple, severe abnormalities, including craniofacial, heart and/or thumb malformations. In contrast, patients with RPL11 and RPS19 mutations who presented with multiple physical abnormalities were uncommon, three patients out of 18, and 16 out of 76, respectively (p=0.02 for RPL5 vs RPL11 and p=0.0047 for RPL5 vs RPS19, FET).

In summary, we identified single mutations in four genes as well as the second mutation in RPS17, suggesting that sequence changes in RPS7, RPS17, RPL36, RPS15, and RPS27A are rare events in DBA. Mutations in RPL5 are associated with multiple physical abnormalities including craniofacial, thumbs and heart anomalies, while thumb malformations are predominantly present in patients carrying mutations in RPL11.