ALBERT

Description: Abstract 1321 Poster Board I-343 Wiskott-Aldrich syndrome is characterized by immune deficiency and thrombocytopenia. There is also a qualitative platelet defect in Wiskott-Aldrich syndrome, typically presenting on aggregometry as a failure of the second wave of aggregation. The pathophysiology of this defect has not been studied in detail. To investigate this phenomenon, we enumerated dense bodies and measured serotonin content in the platelets of 7 patients with Wiskott-Aldrich syndrome. Patients ranged in age from 14 to 45 years old. Five of 7 patients had had splenectomy. In these patients, the platelet count ranged from 120,000/mcL to 521,000/mcL. Platelet counts were 20,000/mcL and 46,000/mcL in the 2 patients who had not had splenectomy. The latter 2 patients had occasional petechiae, as did 2 of the splenectomized patients. Three patients had no clinical evidence of platelet dysfunction. One of these 3 had tolerated abdominal surgery without untoward bleeding. Blood specimens were taken in the course of routine follow-up, when patients were otherwise well. Platelets were prepaed for electron microscopy by the whole mount technique. For serotonin content, platelets were counted and lysed in distilled water. Serotonin in the lysate was chemically acetylated and then measured by ELISA. Serotonin levels were normalized to ng of serotonin per 10 9 platelets. The average ± standard deviation dense body count in our patient group was 1.1 ± 0.4 dense bodies per platelet (normal count 6-8 dense bodies/platelet). Serotonin content was measured in three patients, who showed 273 ± 103 ng serotonin per 10 9 platelets (normal value 621-1064 ng/10 9 platelets). Our results are the first quantitative analysis of platelet dense body counts and serotonin level in patients with Wiskott-Aldrich syndrome. In addition to further elucidating the pathophysiology of this disease, these data have implications for understanding the role of the Wiskott-Aldrich syndrome protein in the genesis of dense bodies. Disclosures No relevant conflicts of interest to declare.

Description: Background: In addition to NBEAL2, a single dominant-negative mutation in the GFIB1 gene has been associated with grey platelet syndrome but no association has been mentioned with dense granule deficiency. Here we describe a child with thrombocytopenia, alpha-delta granule deficiency, and a homozygous missense mutation in GFIB1. Case Report:The patient is an 8y/o Hispanic male born to non-consanguineous parents. Prenatal and birth history were unremarkable. No family history of blood disorders or pediatric malignancies. The boy has 2 healthy older siblings, no dysmorphic features, and normal skin pigmentation and eye findings. Renal function and hearing are normal. At 3mo he was noted to have multiple spontaneous petechiae along with an isolated thrombocytopenia of 46K/uL. At 10 months, a bone marrow evaluation showed increased megakaryocytes suggestive of ITP. By 3years of age, he received three treatments of IVIG without an adequate response. His platelet counts have generally ranged between 30-50K/uL. With acute illnesses, they drop to 15-20K/uL. His bleeding symptoms have primarily been spontaneous bruising and petechiae as well as prolonged epistaxis. His symptoms have been generally controlled with anti-fibrinolytic agents alone. Platelet transfusions have been reserved for surgical procedures or significant bleeding symptoms. Further evaluations over the past 5 years have included peripheral smears, showing atypical large hypo-granular platelets, and two additional bone marrow aspirates, showing megakaryocytic hyperplasia with numerous osteoclast-like forms and occasional small mono-lobated megakaryocytes and evident emperipolesis. Anti-nuclear antibody testing and platelet direct and indirect antibody testing were negative. Platelet electron microscopy showed that platelets virtually contained no dense granules (0.05 dense granules/platelet, 200 platelets) and about 30-40% of the platelets had markedly decreased alpha granules. Some platelets had complex canalicular networks and membrane vacuoles. These features are consistent with an alpha-delta platelet storage-pool deficiency. A myeloid malignancy mutation panel, performed using next generation sequencing, detected no variants of known significance. Gene sequencing for MHY9-related disorders as well as DNA breakage analysis for Fanconi's anemia were negative. However, DNA sequencing of the patient's sample revealed a homozygous missense mutation (c923 T〉C; p.Leu308Pro) in the GFIB1gene; each parent carried one copy of this change. Conclusion: GFIB1, mapped to chromosome 9q34.13, encodes a transcriptional repressor that is important for megakaryopoiesis. It has been reported in patients with gray platelet syndrome; an inherited platelet disorder associated with thrombocytopenia and decreased alpha granules. This case points to an association of biallelic GFIB1 mutations with alpha-delta granule deficiency. This case underscores the importance of platelet esoteric testing and molecular analysis in the diagnosis of hereditary platelet disorders. As a targetable mutation in the future, this could revolutionize the early diagnosis and treatment of this rare platelet disorder. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 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

Description: Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

Description: Key Points Small deletions in the RPS14 region of 5q must be considered in atypical 5q− syndrome and nonclassical Diamond Blackfan anemia.

Description: Abstract 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.

Description: Abstract 1168 Background: DBA is a congenital anemia that results from failure of adequate erythrocyte expansion from erythroid precursors, often associated with congenital physical abnormalities. Mutations of eleven ribosomal protein (RP) genes have been confirmed in association with DBA, with several other RP gene changes of uncertain significance reported in isolated cases. However, despite a series of sequencing studies in DBA including all of the RP genes, no genetic abnormality has been identified in ∼40% of patients, suggesting the possibility of additional genetic changes. Aim: We performed SNP array genotyping on DBA blood samples without identified RP gene mutations to determine whether allelic loss of one or more RP genes, which would not be identified by sequencing, might be responsible for DBA in these patients. Method: We performed SNP array genotyping on 23 DBA probands, 5 parents, 1 affected and 1 unaffected sibling. Genomic DNA from peripheral blood mononuclear cells was hybridized to HumanOmni1-Quad BeadChips and copy number variants (CNVs) were identified using a hidden Markov model-based algorithm integrating signal intensity with SNP distribution. Regions of putative copy variation were queried for overlap with RP genes. Fractional mosaicism was estimated by modeling B-allele frequency data against mosaic models using an iterated, non-linear continuous distribution function regression model. Results: We identified regional monosomy of chromosomal regions containing RP genes with established relevance to DBA in 3 probands: 1) a 16 Kb deletion involving RPS26, 2) a 1.6 Mb deletion that includes RPS17, and 3) an 828 Kb deletion involving RPS19. The latter patient had macrocephaly with developmental delay and was not steroid responsive. The former two patients were steroid-responsive and lacked physical abnormalities. In addition to constitutional copy loss at RP gene loci, we identified 3 examples of mosaic regional copy loss involving RP genes. Mosaicism is visualized by SNP genotyping as a symmetric splitting around 0.5 of the B-allele frequency plot into two distinct histogram peaks in regions of reduced signal intensity (Fig 1a & b). One patient, discussed in detail separately, demonstrated two discrete regions of mosaic loss on 5q in a region that includes RPS14 and is associated with 5q- MDS (Fig 1a). The monosomic fraction was estimated at 64% in mixed peripheral blood DNA. Similar analysis of DNA from sorted lymphoid and myeloid peripheral blood populations demonstrated near total monosomy in myeloid and disomy of these regions in lymphoid DNA. This abnormality was not found in either parent, further confirming somatic copy loss. Two siblings evaluated from a family of 3 affected siblings demonstrated variable mosaicism of 3q, including the telomeric region containing RPL35a (Fig 1b). The degree of deletion was greatest at the telomeric end (23% at 187M-qter) and decreased approaching the centromere. DNA from a third sibling and father were not available; however no similar abnormality was detected in the mother. All siblings have modest neutropenia but lack physical abnormalities. None responded to corticosteroid therapy though two achieved spontaneous remission at 12 and 16 years of age. The father had a history of anemia and developed MDS progressing to AML at 44 years of age. A third variably mosaic chromosomal abnormality was identified on the long arm of chromosome 15 in one proband. The involved region spans the entire long arm, and like the 3q abnormality, shows the highest fractional mosaicism at the telomere. This patient is also steroid-unresponsive and lacks physical abnormalities. RPS17, RPL4, RPLP1 and RPS27L all lie in the telomeric half of 15q. Conclusion: We detected deletions of known or suspected DBA-related genes in a cohort of patients in whom no mutations could be found by RP sequencing. The rate of detection (6/23, 26%) suggests that deletions may explain a portion of patients in whom RP gene mutations cannot be identified. These data demonstrate the novel finding of chromosome-specific variable mosaicism in a hematologic disorder, a finding which suggests inheritance (3q-) or potentially acquisition (15q-) of a factor predisposing to regional chromosomal instability. Finally, these findings suggest the possibility of hematopoietic mosaicism as a determinant of remission from anemia seen in ∼20% of patients with DBA. Disclosures: No relevant conflicts of interest to declare.