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Diamond Blackfan anemia (DBA) is predominantly an autosomal dominant inherited red cell aplasia primarily caused by pathogenic germline variants in ribosomal protein genes. DBA due to pathogenic RPL35A variants has been associated with large 3q29 deletions and phenotypes not common in DBA. We conducted a multi-institutional genotype-phenotype study of 45 patients with DBA associated with pathogenic RPL35A germline variants and curated the variant data on 21 additional cases from the literature. Genotype-phenotype analyses were conducted comparing patients with large deletions versus all other pathogenic variants in RPL35A. Twenty-two of the 45 cases had large deletions in RPL35A. After adjusting for multiple tests, a statistically significant association was observed between patients with a large deletion and steroid-resistant anemia, neutropenia, craniofacial abnormalities, chronic gastrointestinal problems, and intellectual disabilities (p

Background Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. Procedure Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). Results This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. Conclusions L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

Diamond Blackfan anemia (DBA) is predominantly an autosomal dominant inherited red cell aplasia primarily caused by pathogenic germline variants in ribosomal protein genes. DBA due to pathogenic RPL35A variants has been associated with large 3q29 deletions and phenotypes not common in DBA. We conducted a multi-institutional genotypephenotype study of 45 patients with DBA associated with pathogenic RPL35A germline variants and curated the variant data on 21 additional cases from the literature. Genotype-phenotype analyses were conducted comparing patients with large deletions versus all other pathogenic variants in RPL35A. Twenty-two of the 45 cases had large deletions in RPL35A. After adjusting for multiple tests, a statistically significant association was observed between patients with a large deletion and steroid-resistant anemia, neutropenia, craniofacial abnormalities, chronic gastrointestinal problems, and intellectual disabilities (P

Objective Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome. The mainstays of treatment involve chronic red cell transfusions, long-term glucocorticoid therapy, and stem cell transplantation. Systematic data concerning endocrine function in DBA are limited. We studied patients in the DBA Registry (DBAR) of North America to assess the prevalence of various endocrinopathies.

Diamond Blackfan Anemia Syndrome (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, most often diagnosed during infancy. Congenital anomalies and predisposition to cancer are also important features of DBA. Establishment of a molecular diagnosis in a patient with DBA is critical to determine treatment strategies (i.e. the identification of compatible related transplant donors), as well as developing reproductive strategies for genetically at risk families. The overwhelming majority (>98.75%) of DBA patients with a molecular diagnosis have mutations in a Ribosomal Protein (RP) gene. Targeted and exome sequencing (WES) strategies can identify RP mutations in >70% of DBA patients (Ulrisch et al. Am J Hum Genet. 2018). Single Nucleotide Polymorphism Comparative Genome Hybridization (SNP array) detects >30 kb deletions of RP genes (which cannot be identified by sequencing) in ~10% of DBA patients (Farrar et al. Blood. 2011), leaving ~20% of DBA patients without a molecular diagnosis. We hypothesized that smaller copy number variants (CNVs - either insertions or deletions) in RP genes that are below the limit of detection of SNP array are responsible for the remaining 20%. To test this hypothesis we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 6 patients who had no mutations detected by WES or SNP array. On average, we aligned ~1x1010paired end reads of 250 base pairs for each patient (~83X coverage of the genome). The aligned sequences were analyzed for CNVs using two independent software packages. Delly analyzes the two ends of each sequence read and maps them to the current human reference genome. Read ends that map further apart than expected are flagged as potential CNVs. CNVkit estimates regions of copy loss by changes in average sequencing depth. Using relatively relaxed thresholds in Delly and CNVkit we identified ~100 candidate CNVs in each patient. We filtered out CNVs present in public databases and focused on those CNVs in the region of the RP genes. This analysis identified 2-5 potential RP gene associated CNVs in each patient. We designed PCR primers that flanked each putative CNV and confirmed at least one RP CNV in all 6 patient DNAs. At this time, the CNVs in two patients are in the process of evaluation. We have validated causative RP CNVs in the other 4 patients, representing one known and three novel DBA genes. One patient had a 464 bp deletion in 3rdintron of the RPL27 gene, which is mutated in other DBA patients. We hypothesized that the deletion caused a splicing defect. Using a mini gene in which the second intron of the gamma globin gene was replaced with the 3rdintron of either the wild type or mutant RPL27 gene, we showed that the mutant exon was not spliced. An alternative hypothesis, that the deletion removed an enhancer element, was also tested, but no enhancer activity was detected. We conclude that the RPL27exon deletion causes aberrant splicing leading to an unstable RPL27 mRNA and haploinsufficiency of RPL27. A second patient had a 3.5 kb deletion at the 3' end of the RPS5 gene, including the stop codon and poly A addition site. We hypothesized that the lack of the 3' processing signals would lead to an unstable mRNA. To test this hypothesis we generated MYC and FLAG tagged wildtype and 3' deleted RPS5 genes and co-transfected them into 293T cells. Regardless of the tag used, RT-PCR analysis showed a severe reduction in the mutant mRNA levels. Western Blot analysis demonstrated that only the wild type protein was expressed, leading to the conclusion that the RPS5 truncation led to an unstable RPS5 mRNA and haploinsufficiency of RPS5. A third patient had a 28 kb deletion that removes the RPS9 gene. shRNA knockdown of RPS9 mRNA in normal CD34+ cells inhibited erythroid differentiation, leading to the conclusion that RPS9 deficiency causes DBA. Finally, we observed a 3 bp insertion in exon 6 of the RPL14 gene. The deletion adds an alanine residue to a string of 10 alanines in the wild type allele. We confirmed the insertion by targeted sequence analysis of patient DNA. Our data show that WGS can identify small CNVs that cause DBA in at least 2/3 of patients who do not have a mutation detectable by other methods. We believe that WGS analysis following targeted sequencing, SNP array and WES can identify virtually all DBA mutations. With declining WGS costs, we recommend adding WGS to the molecular diagnostic pipeline for genetic testing of DBA. Disclosures Farrar: Novartis: Research Funding. Vlachos:Novartis: Other: Steering committee member.

Background: Diamond Blackfan anemia (DBA) is a congenital anemia characterized by failure of adequate erythrocyte expansion from hematopoietic precursors. The genetic basis of DBA is largely established, with mutation or deletion of at least 19 structural ribosomal protein (RP) genes, a RP chaperone (TSR2), or a pivotal erythroid transcription factor (GATA1) identifiable in most DBA cases. However, the marked clinical variability in DBA-including varying ages of presentation, severity of anemia, responsiveness to corticosteroids, and sporadic hematologic remissions-remains unexplained by genotype and may be modulated by epigenetic factors. Further understanding of this variability is of potential therapeutic relevance for biomarkers of steroid response and remission as well as in application to novel treatment approaches. Aim: We characterized genome-wide methylation and chromatin accessibility of erythroid progenitors from normal controls and DBA patients during erythroid commitment in order to identify the epigenetic features associated with erythroid failure in DBA, steroid response, and remission. Methods: We expanded and sorted CD71+/CD235- (transferrin receptor/glycophorin A) and CD71+/CD235+ erythroid cell fractions from DBA patients and controls after isolation of primary circulating CD34+ cells from peripheral blood (O'Brien et al, Blood 129(23):3111, 2017). We performed DNA methylation analysis using Illumina Epic arrays in 9 control and 22 DBA subjects (11 transfusion-dependent, 6 steroid responsive, 5 remission), characterizing differentially methylated probes and regions among groups. To define broad chromatin domains, we identified A/B chromatin compartments (representing open/closed chromatin) using long-range correlations in methylation data as previously described (Fortin et al. Genome Biol 16:180, 2015). To identify discrete local changes in chromatin accessibility, we performed ATAC-sequencing in 9 controls and 17 DBA patients (10 transfusion, 6 steroid, 1 remission). Results: Global DNA methylation falls during erythroid commitment, with 258,618 differentially hypermethylated CpG sites in normal control GlyA- cells compared to their more differentiated GlyA+ counterparts. This pattern is exaggerated in DBA samples, with 297,926 sites hypermethylated in GlyA- cells. We identified 17,392 CpC sites that distinguish GlyA- DBA progenitors from normal progenitors (16,953 hyper- and 439 hypomethylated). We identified 1,749 differentially methylated sites in comparison of transfusion dependent and remission DBA, as well as 79 sites different between transfusion dependent and steroid responsive DBA. Using genome-wide methylation data, we evaluated A/B compartment organization among these groups to identify large regions of open and closed chromatin during normal and DBA early erythroid differentiation. We observe significant shifts in A/B compartments in normal cells concurrent with the acquisition of GlyA surface expression. At genome scale, transfusion dependent and steroid resistant DBA samples are generally similar to each other, with thousands of regions where A/B identity are closely matched in DBA, but diametrically opposed to the configuration in stage-matched normal controls. Intriguingly, remission samples generally matched A/B compartments of other DBA samples in GlyA- fractions but GlyA+ compartments more closely resemble those of the controls, indicating that normalization of chromatin structural maturation accompanies hematologic remission. We generated a uniform set of 8,877 enriched ATAC-seq peaks on autosomes for differential chromatin accessibility analysis. As with methylation data, a large proportion (25%; 1085 up and 1114 down, B-H adj. P < 0.1) showed differential accessibility in normal control GlyA- vs GlyA+ cells. Steroid-responsive cases showed additional regions of differential accessibility during early maturation, with 31% of regions (1515 up and 1248 down) differentially accessible. Among transfusion dependent DBA patients, this count was much higher, with over half of peak regions (52%, 2400 up and 2216 down, B-H adj. P < 0.1) showing differential accessibility. Conclusion: Epigenetic maturation is broadly altered in DBA erythroid progenitors compared to stage matched normal controls, with specific changes identifiable in patients responding to steroids and in remission. Disclosures No relevant conflicts of interest to declare.

Diamond Blackfan anemia (DBA) is a rare, inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. The patients usually present during infancy or early childhood, but can also present in adulthood. In the majority of cases DBA is due to a mutation in a small or large ribosomal protein (RP) subunit leading to RP haploinsufficiency. The only treatments for the anemia of DBA are red cell transfusions (accompanied by iron chelation), oral corticosteroid therapy or stem cell transplantation. Pospisilova et al. (Haematologica 2007; 92(5):e66-67) reported one complete and two partial erythroid responses after the use of the branched chain amino acid L-leucine in 6 select patients. In skeletal muscle, leucine supplementation can upregulate components of the protein synthetic machinery, including ribosomal proteins, promoting protein translation. Mouse and fish models of DBA respond with amelioration of anemia to L-leucine. We therefore proposed to study the effect of L-leucine on transfusion dependence and growth in subjects with DBA. Methods: The primary objectives were to determine the feasibility of administering L-leucine in subjects with DBA who are red cell transfusion-dependent and to determine the efficacy of L-leucine to produce a hematologic and growth response. The secondary objective was to determine the safety profile of L-leucine. Twelve study sites were involved in this multi-center, Phase I/II study with an anticipated accrual of 50 subjects. A dose of 700 mg/M2 orally three times per day for 9 months was used. Inclusion criteria included age > 2 years, the diagnosis of DBA and transfusion dependence with adequate kidney and liver function. Response was evaluated at 9 months with Complete Response (CR) defined as no further transfusions required and Hb >9; Partial Response (PR): Hb < 9 gm/dL with an increase in reticulocyte count and transfusion interval; and No Response (NR): no change in transfusion needs, Hb or reticulocyte count . Growth percentiles were evaluated at baseline and at completion of 9 months of treatment and the growth velocity change was calculated. Results: The study opened July 2014 and closed February 2017; 55 subjects were consented; 12 were screen failures; 43 subjects were evaluable. There were 21 males; the median age was 9 years 1 month (2 years 5 months - 46 years 1 month). There were no untoward side effects experienced by any subject that were attributable to the L-leucine. Two subjects had an erythroid CR and 1 subject had a PR. The CRs occurred at 1 month and 3 months after start of L-leucine. The subject with PR had an elevated reticulocyte count but was not able to maintain a Hb >9 gm/dL without a transfusion and thus was not transfusion independent. Of the 30 subjects with growth potential who received L-leucine 10 experienced a positive growth velocity change at 9 months of therapy compared to baseline. At a median age of 7.5 years, the mean pre-leucine height percentile was 27 +/- 17.9 and the post-leucine height percentile was 35 +/- 19.9 (p Conclusions: The administration of L-leucine is safe. L-leucine administration resulted in an erythroid response in 7% of subjects and an increased growth velocity in 33% of growing subjects. Based upon extrapolation from animal models, it is likely that this dose was suboptimal. We hypothesize that higher doses of L-leucine will lead to hematologic responses in more subjects who are transfusion dependent. The potential benefit of added growth in children with DBA may improve final adult height. Disclosures Glader: Agios: Consultancy, Research Funding.

Background: RPL35A, a gene encoding a large ribosomal subunit protein located at the telomeric end of chromosome 3q (3q29-qter) is essential for rRNA processing, ribosomal biogenesis, cell proliferation, and apoptosis, and accounts for a subset of patients with Diamond Blackfan anemia (DBA). Reported pathogenic RPL35A mutations include single-nucleotide variants (SNVs), small insertion/deletions (indels), and large contiguous gene deletions associated with 3q29 microdeletion syndrome. 3q29 deletion syndrome is an overlapping syndrome that consists of developmental and intellectual disability with or without dysmorphic features, and other congenital anomalies but no anemia or cytopenia. The clinical phenotype and disease severity of patients with RPL35A-related DBA may be influenced by other genes deleted within 3q29 and be different in patients with large deletions compared to those with SNVs or small indels. Objectives: To determine whether DBA patients with large deletions of the 3q29 region have a more severe disease phenotype than those with SNVs or small indels in RPL35A, and whether other genes deleted within the 3q29 region might contribute to some of the features. Methods and Results: We identified 40 patients in a multi-institutional, international collaborative study of patients with DBA with RPL35A haploinsufficiency: 21 had deletion of RPL35A as part of 3q29 contiguous gene deletion, ranging in size from 0.012 Mb to 11 Mb in 16 patients; the extent of the deletion beyond RPL35A in either direction was unknown in 5 patients. Nineteen patients had SNVs or small indels (7 missense, 1 nonsense, 3 splice site, 6 indels and 2 unclear pathogenicity). Thirty-nine of 40 patients had severe anemia, 32 had neutropenia at some time and 3 had thrombocytopenia. Compared to the patients with SNVs or small indels, a significantly higher proportion of patients with RPL35A haploinsufficiency due to 3q29 deletion had steroid-resistant anemia (17 vs 7; p=0.009), severe chronic or intermittent neutropenia requiring treatment with G-CSF (7 vs 0; p=0.009), and humoral and/or cellular immunodeficiency (7 vs 1; p=0.046) diagnosed in some patients due to recurrent infections requiring hospitalizations (10 vs 2; p=0.03). Learning difficulties (12 vs 2; p=0.003), craniofacial abnormalities (11 vs 3; p=0.02), skeletal and limb defects (9 vs 2; p=0.03) or multiple physical anomalies (≥3) were also more frequent in patients with large deletions than in those with SNVs or indels (11 vs 3; p=0.02). Microcephaly (28%), short stature (33%), cardiac defects (28%) and/or urogenital abnormalities (23%) were equally distributed. The potential genes of interest near RPL35A that may be associated with immune dysregulation and/or neutropenia are RNF168, TFRC, PAK2, PIGZ, DLG1 and LMLN. One or more of these genes were deleted in at least 6 of 7 patients with neutropenia or immunodeficiency. Eight of 9 patients with malformations involving extremities, skeleton and ribs had deletions of TCTEX1D2 which is associated with rib/thoracic dysplasia and polydactyly. The genes of interest deleted in patients with developmental delay and learning disabilities included PAK2 and DLG1 in 9 patients, as well as RNF168, PPP1R2, TNK2 and q29 KIAA0226 in 8 of 12 patients in whom the extent of the deletion was known. Summary and Conclusion: Patients with DBA due to RPL35A contiguous gene deletions are clearly different from those with SNVs or small indels and have increased frequency of steroid-resistant transfusion-dependent anemia, severe neutropenia, immunodeficiency, learning/developmental delay, and craniofacial/skeletal/limb anomalies. Distinction of this subtype of DBA with RPL35A haploinsufficiency due to 3q29 contiguous gene deletion is relevant to their management, and evaluations should include a work-up for immunodeficiency. Further studies are needed to determine whether the complex phenotypes and severe disease manifestations in these patients are solely due to RPL35A haploinsufficiency or to the potential effect of other genes deleted in the 3q29 region. Genotype-phenotype characterization and comparison of DBA patients with 3q29 deletion with those due to deletions in other ribosomal protein genes may determine similarities or differences in disease phenotypes related to large gene deletions versus the influence of multigenic contiguous deletions. Supported by: AZV 16-32105A Disclosures Kattamis: Novartis: Consultancy, Honoraria; CELGENE: Consultancy, Honoraria; ApoPharma: Honoraria; Vifor Pharma: Consultancy. Niemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome. The mainstays of treatment involve chronic red cell transfusions, long-term glucocorticoid therapy, and stem cell transplantation. Systematic data concerning endocrine function in DBA are limited. We studied patients in the DBA Registry (DBAR) of North America to assess the prevalence of various endocrinopathies.In a pilot study, retrospective data were collected for 12 patients with DBA. Subsequently, patients with DBA aged 1-39 years were recruited prospectively. Combined, 57 patients were studied; 38 chronically transfused, 12 glucocorticoid-dependent, and seven in remission. Data were collected on anthropometric measurements, systematic screening of pituitary, thyroid, parathyroid, adrenal, pancreatic, and gonadal function, and ferritin levels. Descriptive statistics were tabulated and group differences were assessed.Fifty-three percent of patients had ≥ 1 endocrine disorder, including adrenal insufficiency (32%), hypogonadism (29%), hypothyroidism (14%), growth hormone dysfunction (7%), diabetes mellitus (2%), and/or diabetes insipidus (2%). Ten of the 33 patients with available heights had height standard deviation less than -2. Low 25-hydroxy vitamin D (25(OH)D) levels were present in 50%. A small proportion also had osteopenia, osteoporosis, or hypercalciuria. Most with adrenal insufficiency were glucocorticoid dependent; other endocrinopathies were more common in chronically transfused patients.Endocrine dysfunction is common in DBA, as early as the teenage years. Although prevalence is highest in transfused patients, patients taking glucocorticoids or in remission also have endocrine dysfunction. Longitudinal studies are needed to better understand the etiology and true prevalence of these disorders.

Summary 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.

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.