Diamond-Blackfan anemia (DBA) is characterized by red cell failure, the presence of congenital anomalies, and cancer predisposition. In addition to being an inherited bone marrow failure syndrome, DBA is also categorized as a ribosomopathy as, in more than 50% of cases, the syndrome appears to result from haploinsufficiency of either a small or large subunit-associated ribosomal protein.
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
• Small deletions in the RPS14 region of 5q must be considered in atypical 5q2 syndrome and nonclassical Diamond Blackfan anemia.Classical 5q2 syndrome is an acquired macrocytic anemia of the elderly. Similar to Diamond Blackfan anemia (DBA), an inherited red cell aplasia, the bone marrow is characterized by a paucity of erythroid precursors. RPS14 deletions in combination with other deletions in the region have been implicated as causative of the 5q2 syndrome phenotype. We asked whether smaller, less easily detectable deletions could account for a syndrome with a modified phenotype. We employed singlenucleotide polymorphism array genotyping to identify small deletions in patients diagnosed with DBA and other anemias lacking molecular diagnoses. Diminutive mosaic deletions involving RPS14 were identified in a 5-year-old patient with nonclassical DBA and in a 17-year-old patient with myelodysplastic syndrome. Patients with nonclassical DBA and other hypoproliferative anemias may have somatically acquired 5q deletions with RPS14 haploinsufficiency not identified by fluorescence in situ hybridization or cytogenetic testing, thus refining the spectrum of disorders with 5q2 deletions. (Blood. 2013;122(14):2487-2490) IntroductionDiamond Blackfan anemia (DBA) is a rare, inherited 1,2 red cell aplasia and cancer predisposition syndrome 3 presenting at a median age of 2 months with macrocytic anemia, marrow erythroid hypoplasia, and reticulocytopenia. 4 It is associated with elevated erythrocyte adenosine deaminase (eADA) activity in 80% of patients.5 Approximately 65% of patients have a mutation or deletion in 1 of 10 genes encoding ribosomal proteins (RP), resulting in RP haploinsufficiency. 1,[6][7][8][9] In contrast to DBA, the 5q2 syndrome, a subtype of myelodysplastic syndrome (MDS), is an acquired red cell failure disorder associated with interstitial deletions of chromosome 5q, predominantly diagnosed in elderly women.10 Ebert et al showed that the erythroid defect in 5q2 syndrome is caused by the monoallelic loss of RPS14, 11 establishing most DBA and the 5q2 syndrome as ribosomopathies.Here we describe 2 patients, 1 diagnosed with DBA and 1 with myelodysplastic syndrome, who were found to have acquired, clonal abnormalities with smaller than previously reported deletions of 5q, which led to a successful treatment intervention in one of them. Study design SNP array genotypingSingle-nucleotide polymorphism (SNP) array genotyping was performed on whole-blood and lineage-specific DNA from 105 patients from the DBA Registry and 1 patient referred with an atypical red cell aplasia, as previously described. 7 Estimation of mosaic monosomyRegions of mosaic monosomy were identified by visual inspection of minor allele frequency plots. A normal continuous distribution function (CDF) of the heterozygous minor alleles was averaged from 5 dizygous regions for each array analyzed. The normal CDF was fitted to the empirical CDF of the mosaic region to estimate fractional mosaicism, as previously described. 12 Accession numbe...
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 <0.01). The mean weight percentile pre-leucine was 35.4 +/- 26.6 versus the post-Leucine weight percentile of 38.4 +/- 28.1. 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.
LBA-2 Background: 5q- myelodysplastic syndrome is a rare, acquired macrocytic anemia with a female predominance. The bone marrow is characterized by a paucity of erythroid precursors with relatively normal leukocyte and platelet counts and no excess blasts. The mean age at diagnosis is approximately 70 years. The phenotype of 5q deletion has been shown to result from haploinsufficiency of the RPS14 gene. Historically red blood cell transfusions have been the primary treatment; however lenalidomide has recently been effective in ameliorating the anemia with a response rate of 67%. DBA is a rare heritable red cell aplasia which usually presents in infancy. It too is characterized by a bone marrow deficient in erythroid precursors. Mutations or deletions in eleven ribosomal protein (RP) genes, resulting in protein haploinsufficiency, have been reported in 50–60% of patients. To date RPS14 mutations have not been identified in DBA patients. Array Comparative Genomic Hybridization (CGH) has been used to identify large deletions in patients with DBA, but a more sensitive approach was hypothesized to identify additional deletions. Purpose: To address the question of whether chromosomal deletions could be the underlying defect in patients with DBA who did not have mutations in the known RP genes, Single Nucleotide Polymorphism (SNP) genotyping array hybridization was utilized. Methods: Seventy-five patient samples from the DBA Registry (DBAR) underwent resequencing of 80 RP genes. Approximately 40% of the patients had no identifiable mutation. High resolution SNP array genotyping analysis was done on 23 probands who did not have a mutation detected by resequencing. Results: An acquired internal deletion on chromosome 5q involving RPS14 was identified in one of 23 patients with presumed DBA. The patient presented with anemia at 5 10/12 years of age. The hemoglobin was 8.4 g/dl, MCV 108.2 fL, and reticulocyte count 0.4%. The erythrocyte adenosine deaminase (eADA) activity, elevated in 85% of DBA patients, was normal. The bone marrow showed decreased cellularity and megaloblastoid changes in the erythroid series. There were adequate numbers of megakaryocytes with no hypolobulation. Cytogenetics performed at diagnosis in 1991 appeared normal. The patient had no significant family history or congenital anomalies. A diagnosis of non-classical DBA was made. The patient failed a trial of corticosteroids and had remained transfusion-dependent for 19 years. No RP gene mutation was identified by sequencing. SNP array genotyping analysis identified mosaicism in two discrete regions covering ∼17.7 Mb on 5q-, with an estimated 63.7% monosomy and 36.3% disomy in this region. The major region extends from 141.1M to 157.2M (hg18), including all of the 5q- syndrome commonly deleted region (CDR) at 5q33, though it excludes the 5q31 CDR, miR146a, as well as Cdc25C and PPP2Acα, factors for which haploinsufficient expression has previously been suggested to be important in response to lenalidomide. SNP array genotyping from purified populations indicated that lymphocytes were >95% normal, while the myeloid cells were >95% 5q-. CD34+ cells showed a marked decrease in both myeloid and erythroid colony formation. Patient fibroblasts were normal and neither of the parents have 5q abnormalities by SNP analysis. Although the deletion was not identified in 1991, the 46,XX,der(5)del(5)(q15q22)del(5)(q32q33) deletion was detected on high resolution karyotyping in a post-SNP array genotyping marrow sample. Haploinsufficiency of RPS14 was confirmed by quantitative RT-PCR. After a trial of lenalidomide, complicated by Grade 4 neutropenia and Grade 3 thrombocytopenia, the patient has a reticulocyte count of 7.4% (from a previous baseline of <0.2%) and has achieved a hemoglobin of 11.1 gm/dl without transfusion support. Conclusions: Patients with non-classical DBA, who also have no congenital anomalies and normal eADA activity, may have somatically acquired 5q deletions with RPS14 haploinsufficiency. The DBAR is presently performing SNP array genotyping on other DBA patients who fit these criteria. These data suggest that haploinsufficiency of Cdc25C and PPP2Acα are not required for an erythroid response to lenolidamide. Reclassification of non-classical DBA patients as 5q- MDS offers them a potential treatment option with lenalidomide. Disclosures: No relevant conflicts of interest to declare.
Background: DBA is a rare genetic disorder characterized by faulty ribosome biogenesis, leading to pro-apoptotic erythropoiesis and red cell failure. The Diamond Blackfan Anemia Registry (DBAR) was established in 1993 to provide a robust database for investigating the biology and epidemiology of DBA. The DBAR defines remission in DBA as independence from red cell transfusion or corticosteroid therapy for greater than 6 months. An understanding of factors influencing DBA remission may provide insights into the pathophysiology of DBA and ultimately can lead to improved treatment options. Method: Patients were enrolled in the DBAR with informed consent. Patients who met the remission criteria for this study were evaluated. Patients who reported a remission were contacted and completed a “remission questionnaire”; further information was obtained from the patients’ physicians and verified, where possible, from medical records. Results: Of 555 patients enrolled in the DBAR, 67 patients have experienced a remission (actuarial likelihood approximately 20%). The male: female ratio for remission patients vs. the total DBAR population is 1:0.97 vs. 1:1.03, respectively. Sixty-four percent of patients have congenital anomalies, compared to 47% in the total population; the difference is not statistically significant. All categories of congenital anomalies (e.g. orofacial, cardiac, renal, skeletal, etc.) are represented in the remission population. The median age at diagnosis is 2.9 months (range, 0 to 14.9 years). The majority of patients were started on 2mg/kg/day of prednisone or methylprednisolone at a median age of 3.6 months (range, 4 days to 15.2 years). Sixty-six percent entered remission while on steroid therapy. The median total duration on steroids was 36 months (range, 1 month to 37.6 years) and the median duration from start of steroids to beginning of taper was 3.6 months (range, 1 month to 4.6 years). Seventeen percent of patients went into remission with less than one year of steroids and 55% were in remission within 5 years of start of steroids. Fifteen percent remitted while receiving chronic transfusion therapy. Six percent never received steroids prior to remission and 7.5% of remitters initially responded to steroids but became steroid refractory prior to remission. Median duration of treatment to remission and duration of remission were 16 months (range, 6 months to 38.2 years) and 14 years (range, 1 to 46.8 years) respectively. The median age of remission was 6.4 years (range, 0.8 to 39 years); males 6.5 years (range, 0.8 to 39 years) and females 6.3 years (range, 1.1 to 26 years). Of note, remissions were observed in DBA patients or in affected family members of probands with mutations in 4 of the 6 genes (RPS 19, RPS 24, RPL35a, and RPS11) known to be mutated in DBA as well as in those with no known mutation, suggesting that remission is not restricted to a particular genotype. Additional patients are being genotyped. Conclusion: Remission in patients with DBA is not an uncommon event. Steroid responsiveness is not a prerequisite for remission. There is no obvious phenotypic or genotypic difference between remission and non-remission patients. The expression of a remission phenotype within multiplex families is quite variable. We conclude that remission is not restricted to a particular phenotype or genotype and that the likelihood of remission is influenced by unknown modifier genes and/or epigenetic factors.
4430 Background: Diamond Blackfan anemia is a rare heritable red cell aplasia which usually presents in infancy but can also be diagnosed in childhood and even adulthood. Mutations or deletions in eleven ribosomal protein (RP) genes, resulting in protein haplo-insufficiency have been reported in about 54% of the patients. The 5q- syndrome is an acquired myelodysplastic syndrome (MDS) characterized by a similar erythroid failure. Another RP gene included in the 5q deleted region, RPS14, has been identified as a causal gene in 5q- MDS but has not been reported in DBA. Purpose: Array Comparative Genomic Hybridization has been used to identify large deletions in patients with DBA. This report demonstrates the use of Single Nucleotide Polymorphism (SNP) genotyping array hybridization to identify a patient, previously thought to have DBA, as having a 5q- deletion consistent with 5q- syndrome. Method: Seventy-five patient samples from the Diamond Blackfan Anemia Registry of North America, a patient database of now 608 patients designed to better understand the biology and epidemiology of DBA, underwent resequencing of 80 RP genes. Approximately 40% of the patients had no identifiable mutation. High resolution SNP array genotyping analysis was done on 23 probands from this cohort who did not have a mutation detected in either the resequencing project and/or the targeted sequencing efforts lead by Gazda and colleagues. Result: An acquired internal deletion on chromosome 5q involving RPS14 was identified in one patient with presumed DBA. The patient presented at 5 years 10 months of age with anemia noted on a routine blood count. The hemoglobin was 8.4 grams/dl, MCV 108.2 fL, and reticulocyte count 0.4%. The eADA was normal. The bone marrow showed decreased cellularity and megaloblastic changes in the erythroid series. There were adequate numbers of megakaryocytes with no hypolobulation. The cytogenetics performed at diagnosis in 1991 were reported as normal. The patient had no significant family history of anemia and was found to have no congenital physical anomalies. A diagnosis of non-classical DBA was presumed and the patient failed a trial of corticosteroids. At present the patient has marrow red cell aplasia and is on a chronic transfusion schedule. SNP array genotyping analysis identified mosaicism in two discrete regions covering ~17.7 Mb on 5q-, with an estimated 63.7% monosomy and 36.3% disomy in this region. The major region extends from 141.1M to 157.2M (hg18), including all of the 5q- syndrome commonly deleted region (CDR) at 5q33 though it excludes the 5q31 CDR associated with AML and more aggressive MDS as well as miR146a, a factor recently postulated to play a role in 5q- MDS. SNP array genotyping from purified peripheral blood populations indicated that lymphocytes were greater than 95% normal, while the myeloid cells were greater than 95% 5q-. CD34+ cells obtained from this patient showed a marked decrease in both myeloid and erythroid colony formation when compared with normal cells. Patient fibroblasts were normal and neither of the parents have any 5q anomalies by SNP array genotyping. Although the deletion was not identified in 1991 at the time of the diagnosis, the 46,XX,der(5)del(5)(q15q22)del(5)(q32q33) deletion was able to be detected on high resolution karyotyping in a post-SNP array genotyping marrow sample. Haploinsufficiency of RPS14 was confirmed by quantitative RT-PCR. Conclusion: Patients with non-classical DBA may have unique acquired 5q deletions with RPS14 haploinsufficiency. A search for other acquired somatic mutations or deletions in patients with DBA, in particular non-classical cases, is underway. SNP array genotyping is an essential diagnostic tool in this search. Disclosures: No relevant conflicts of interest to declare.
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