Edyta Niewiadomska

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.

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.

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.

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.

RNA and protein evidence for haplo-insufficiency in Diamond–Blackfan anaemia patients with RPS19 mutations

The genetic basis of Diamond–Blackfan anaemia (DBA), a congenital erythroid hypoplasia that shows marked clinical heterogeneity, remains obscure. However, the fact that nearly one‐quarter of patients harbour a variety of mutations in RPS19, a ribosomal protein gene, provides an opportunity to examine whether haplo‐insufficiency of RPS19 protein can be demonstrated in certain cases. To that end, we identified 19 of 81 DBA index cases, both familial and sporadic, with RPS19 mutations. We found 14 distinct insertions, deletions, missense, nonsense and splice site mutations in the 19 probands, and studied mutations in 10 patients at the RNA level and in three patients at the protein level. Characterization of the mutations in 10 probands, including six with novel insertions, nonsense and splice site mutations, showed that the abnormal transcript was detectable in nine cases. The RPS19 mRNA and protein in CD34+ bone marrow cells identified haplo‐insufficiency in three cases predicted to have one functional allele. Our data support the notion that, in addition to rare DBA patients with the deletion of one allele, the disease in certain other RPS19 mutant patients is because of RPS19 protein haplo‐insufficiency.

Frameshift mutation in p53 regulator RPL26 is associated with multiple physical abnormalities and a specific pre-ribosomal RNA processing defect in diamond–blackfan anemia†

Diamond–Blackfan anemia (DBA) is an inherited form of pure red cell aplasia that usually presents in infancy or early childhood and is associated with congenital malformations in ∼30–50% of patients. DBA has been associated with mutations in nine ribosomal protein (RP) genes in about 53% of patients. We completed a large‐scale screen of 79 RP genes by sequencing 16 RP genes (RPL3, RPL7, RPL8, RPL10, RPL14, RPL17, RPL19, RPL23A, RPL26, RPL27, RPL35, RPL36A, RPL39, RPS4X, RPS4Y1, and RPS21) in 96 DBA probands. We identified a de novo two‐nucleotide deletion in RPL26 in one proband associated with multiple severe physical abnormalities. This mutation gives rise to a remarkable ribosome biogenesis defect that affects maturation of both the small and the large subunits. We also found a deletion in RPL19 and missense mutations in RPL3 and RPL23A, which may be variants of unknown significance. Together with RPL5, RPL11, and RPS7, RPL26 is the fourth RP regulating p53 activity that is linked to DBA. Hum Mutat 33:1037–1044, 2012.

Pearson marrow pancreas syndrome in patients suspected to have Diamond-Blackfan anemia

Pearson marrow pancreas syndrome (PS) is a multisystem disorder caused by mitochondrial DNA (mtDNA) deletions. Diamond-Blackfan anemia (DBA) is a congenital hypoproliferative anemia in which mutations in ribosomal protein genes and GATA1 have been implicated. Both syndromes share several features including early onset of severe anemia, variable nonhematologic manifestations, sporadic genetic occurrence, and occasional spontaneous hematologic improvement. Because of the overlapping features and relative rarity of PS, we hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. Here, we evaluated patient DNA samples submitted for DBA genetic studies and found that 8 (4.6%) of 173 genetically uncharacterized patients contained large mtDNA deletions. Only 2 (25%) of the patients had been diagnosed with PS on clinical grounds subsequent to sample submission. We conclude that PS can be overlooked, and that mtDNA deletion testing should be performed in the diagnostic evaluation of patients with congenital anemia.

Difficulties in diagnosing congenital thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura is a very rare condition, especially its familial, genetically determined type called Upshaw Schulman Syndrome (OMIM ♯274150). The study presents 2 families of patients in which congenital thrombotic thrombocytopenic purpura were diagnosed. Symptoms of the disease, such as thrombocytopenia, microangiopathic hemolytic anemia, and kidney disorders were pronounced with varying degrees of severity in 5 children of various ages from these families. Before the final diagnosis, patients were treated for idiopathic thrombocytopenia, hemolytic anemia, and hemolytic-uremic syndrome, respectively. The study was focused on finding the factors responsible for hemolytic anemia. The activity of a disintegrin and metalloprotease with thrombospondin type 1 motif, 13 (ADAMTS13) and ADAMTS13 antibodies were evaluated and genetic tests were performed. Severe ADAMTS13 deficiency was detected in all affected siblings. The diagnosis of Upshaw Schulman Syndrome was confirmed by molecular testing of the gene encoding the von Willebrand factor cleaving protease ADAMTS13 which revealed compound heterozygosity for 1045C>T (R349C) and 3107C>A (S1036X) in the patients of family 1 and homozygosity for the common mutation 4143insA in the patients of family 2. Regular fresh-frozen plasma transfusions were sufficient to control the disease.

Napadowa zimna hemoglobinuria – niedoceniana przyczyna niedokrwistości hemolitycznej u dzieci ☆ ☆☆

Streszczenie Napadowa zimna hemoglobinuria (NZH) jest rzadko wystepującą postacią niedokrwistości hemolitycznej. Cechuje sie wystąpieniem masywnej hemolizy z hemoglobinurią po ekspozycji na zimno. Jej przyczyną są hemolizyny Donatha-Landsteinera. Przedstawiono historie chorob sześciorga dzieci, u ktorych na podstawie wynikow obserwacji klinicznej i badan serologicznych rozpoznano napadową zimną hemoglobinurie. Po zastosowaniu przetoczen koncentratu krwinek czerwonych i steroidow uzyskano dobry efekt terapeutyczny. W artykule zwrocono uwage na koniecznośc unikania wyziebiania pacjentow z niedokrwistością autoimmunologiczną do czasu wykluczenia zimnych autoprzeciwcial.

The Genetic Landscape of Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 1 in 100,000 to 200,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this genetically heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole exome sequencing (WES). Overall, we identified rare and predicted damaging mutations in likely causal genes for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and in one of 19 previously reported genes encoding for a diverse set of ribosomal proteins (RPs). Using WES exon coverage estimates, we were able to identify and validate 31 deletions in DBA associated genes. We also observed an enrichment for extended splice site mutations and validated the diverse effects of these mutations using RNA sequencing in patientderived cell lines. Leveraging the size of our cohort, we observed several robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. In addition to comprehensively identifying mutations in known genes, we further identified rare mutations in 7 previously unreported RP genes that may cause DBA. We also identified several distinct disorders that appear to phenocopy DBA, including 9 individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain > 5% of DBA cases. Overall, this comprehensive report should not only inform clinical practice for DBA patients, but also the design and analysis of future rare variant studies for heterogeneous Mendelian disorders.