Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in approximately 30%-50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.

Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in Diamond-Blackfan anemia patients.

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 that are present in approximately 30%-50% of patients. The disease has been associated with mutations in four ribosomal protein (RP) genes, RPS19, RPS24, RPS17, and RPL35A, in about 30% of patients. However, the genetic basis of the remaining 70% of cases is still unknown. Here, we report the second known mutation in RPS17 and probable pathogenic mutations in three more RP genes, RPL5, RPL11, and RPS7. In addition, we identified rare variants of unknown significance in three other genes, RPL36, RPS15, and RPS27A. Remarkably, careful review of the clinical data showed that mutations in RPL5 are associated with multiple physical abnormalities, including craniofacial, thumb, and heart anomalies, whereas isolated thumb malformations are predominantly present in patients carrying mutations in RPL11. We also demonstrate that mutations of RPL5, RPL11, or RPS7 in DBA cells is associated with diverse defects in the maturation of ribosomal RNAs in the large or the small ribosomal subunit production pathway, expanding the repertoire of ribosomal RNA processing defects associated with DBA.

Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.

Islands within an island: Population genetic structure of the endemic Sardinian newt, Euproctus platycephalus.

The identification of historic and contemporary barriers to dispersal is central to the conservation of endangered amphibians, but may be hindered by their complex life history and elusive nature. The complementary information generated by mitochondrial (mtDNA) and microsatellite markers generates a valuable tool in elucidating population structure and the impact of habitat fragmentation. We applied this approach to the study of an endangered montane newt, Euproctus platycephalus. Endemic to the Mediterranean island of Sardinia, it is threatened by anthropogenic activity, disease, and climate change. We have demonstrated a clear hierarchy of structure across genetically divergent and spatially distinct subpopulations. Divergence between three main mountain regions dominated genetic partitioning with both markers. Mitochondrial phylogeography revealed a deep division dating to ca. 1 million years ago (Mya), isolating the northern region, and further differentiation between the central and southern regions ca. 0.5 Mya, suggesting an association with Pleistocene severe glacial oscillations. Our findings are consistent with a model of southward range expansion during glacial periods, with postglacial range retraction to montane habitat and subsequent genetic isolation. Microsatellite markers revealed further strong population structure, demonstrating significant divergence within the central region, and partial differentiation within the south. The northern population showed reduced genetic diversity. Discordance between mitochondrial and microsatellite markers at this scale indicated a further complexity of population structure, in keeping with male-biased dispersal and female philopatry. Our study underscores the need to elucidate cryptic population structure in the ecology and conservation strategies for endangered island-restricted amphibians, especially in the context of disease and climate change.

Oxygen dependence of K(+)-Cl- cotransport in human red cell ghosts and sickle cells.

KCC activity in normal human red cells (containing haemoglobin A, HbA, and termed HbA cells) is O2-dependent, being active in oxygenated cells but inactive in deoxygenated ones. The mechanism for O2 dependence is unknown but a role for Hb has been suggested. In this paper, we address two main questions. First, do membrane ghosts prepared from HbA cells retain an O2-sensitive KCC activity? Second, how is the response of KCC to changes in O2 tension altered in sickle cell patients heterozygous for HbS and HbC? We found that substantial Cl(-)-dependent K+ influx, indicative of KCC activity, was present in both pink (5-10% normal Hb complement) and white (no measurable Hb) ghosts when equilibrated with air. KCC responded to deoxygenation in pink ghosts only (86 +/- 10% inhibition, mean+/-S.E.M., n = 3), whilst KCC activity in white ghosts remained high (23 +/- 8% inhibition). Results indicate that pink ghosts retain an O2-dependent KCC activity but that this is lost in white ghosts. Second, HbSC-containing red cells showed sickling (88 +/- 3%) when deoxygenated, together with activation of the deoxygenation-induced cation pathway (Psickle) and the Gardos channel. KCC activity, however, was elevated in oxygenated HbSC cells, but inhibited by deoxygenation. Thus Hb polymerisation and sickling could be dissociated from the abnormal response of KCC to deoxygenation observed in HbS-containing red cells. These preparations provide a useful system with which to study the components involved in O2-sensitive membrane transport and why it is perturbed in certain pathological conditions (such as sickle cell disease and oxidant toxicity).

Ribosomal protein S24 gene is mutated in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.

Two-phase culture in Diamond Blackfan anemia: localization of erythroid defect.

The erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell, but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system, we have demonstrated a consistent defect in DBA, regardless of clinical severity, including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)-free phase 1, but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells, and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone, Epo sensitivity was comparable to normal, but erythroid expansion remained subnormal. In clonogenic phase 2 cultures, the number of colonies did not significantly differ between normal cultures and DBA, in the presence or absence of dexamethasone, and at both low and high Epo concentrations. However, colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA, and the defect lies down-stream of the Epo receptor, influencing survival and/or proliferation of erythroid progenitors.

Diamond Blackfan anaemia in the UK: clinical and genetic heterogeneity.

A detailed family study was undertaken of patients notified to the UK Diamond Blackfan Anaemia (DBA) Registry. RPS19 mutations were detected in 16 of 104 families, including two patients with deletions detected by intragenic loss of heterozygosity of tightly linked polymorphisms. In two further cases, polymorphisms were used to determine the parental allele of origin of RPS19 point mutations. A review of clinical details of patients with mutations and patients in the literature having identical or equivalent mutations revealed evidence for a genotype:phenotype correlation with respect to the prevalence of physical anomalies, and the occurrence of mild or variable haematological severity. Nine of 60 patients had a known family history of DBA. Haematological abnormalities, including raised red cell adenosine deaminase activity, were found in first-degree relatives of 16 of 51 (31%) of patients not previously considered to have familial DBA. Results of both parents and any siblings were normal in only 35 of 60 (58%) of cases, who were therefore assumed to have sporadic de novo DBA. The classical inheritance pattern for DBA is autosomal dominant; however, 12 of 60 families (20%) had more than one affected child despite normal results in both parents. These results have important implications for genetic counselling, and for the selection of potential sibling bone marrow donors.

Favorable effect on acute and chronic graft-versus-host disease with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies for marrow transplantation from HLA-identical sibling donors for acquired aplastic anemia.

Between August 1989 and November 2003, 33 patients at our center with acquired aplastic anemia underwent bone marrow transplantation (BMT) from HLA-identical sibling donors with cyclophosphamide and in vivo anti-CD52 monoclonal antibodies (MoAb) for conditioning. The median age at BMT was 17 years (range, 4-46 years). Before BMT, 58% were heavily transfused (>50 transfusions), and 42% had previously experienced treatment failure with antithymocyte globulin-based immunosuppressive therapy. Unmanipulated bone marrow was used as the source of stem cells in all patients except 1. Graft-versus-host disease (GVHD) prophylaxis was with cyclosporine alone in 19 (58%) patients; 14 received anti-CD52 MoAb in addition to cyclosporine. The conditioning regimen was well tolerated without significant acute toxicity. Graft failure was seen in 8 patients (primary, n = 4; secondary, n = 4). Of those whose grafts failed, 4 survived long-term (complete autologous recovery, n = 2; rescue with previously stored marrow, n = 1; second allograft, n = 1). The cumulative incidence of graft failure and grade II to IV acute and chronic GVHD was 24%, 14%, and 4%, respectively. None developed extensive chronic GVHD. With a median follow-up of 59 months, the 5-year survival was 81% (95% confidence interval, 68%-96%). No unexpected early or late infectious or noninfectious complications were observed. We conclude that the conditioning regimen containing cyclophosphamide and anti-CD52 MoAb is well tolerated and effective for acquired aplastic anemia with HLA-matched sibling donors. The favorable effect on the incidence and severity of GVHD is noteworthy in this study and warrants further investigation.