ALBERT

Description: Shwachman-Diamond syndrome (SDS), an autosomal recessive disorder, is characterized by bone marrow dysfunction, exocrine pancreatic insufficiency, congenital abnormalities, and leukemia predisposition (Myers et al., 2012). Most patients with SDS harbor biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is known to play a role in ribosome biogenesis by enabling eviction of the ribosome anti-association factor eIF6 from the 60S ribosomal subunit, to allow formation of the 80S ribosome (Wong et al., 2011). SBDS-depleted cells are, therefore, defective in ribosome assembly. In addition, absence of SBDS sensitizes cells to ultraviolet irradiation, translation inhibitors, and endoplasmic reticulum (ER) stressors, such as tunicamycin (Ball et al., 2009). A recent report indicated that lymphoblastoid cell lines (LCLs) derived from two SDS patients accumulated more DNA damage after being exposed to ionizing radiation (IR) (Morini et al., 2015). A deficiency in DNA repair was alluded to as a possible cause, however, the mechanism underlying this previously unreported phenotype was not determined. In this study, we investigated LCLs derived from five SDS patients with biallelic SBDS mutations and found all to be hypersensitive to IR in a colony survival assay. In this assay, increasing doses of IR resulted in a significantly lower survival fraction in SDS-compared to control-LCLs. We found SBDS expression to increase in control-cells when stressed with IR, suggesting that SBDS is a stress response protein and its absence in SDS-LCLs induces hypersensitivity to IR. Because knockdown of SBDS in HEK293 cells induces an ER stress response (Ball et al., 2009), we examined the expression of the ER stress response factor phospho-eIF2α in untreated and IR exposed SDS-LCLs and found phospho-eIF2α expression to be markedly increased compared to controls. This result indicated that SDS-LCLs may have an activated ER stress response, as was further confirmed by exposing these cells to additional ER stressors, tunicamycin and H2O2, and observing a similar upregulation of phospho-eIF2α. Because ER stress is known to suppress DNA double strand break (DSBR) (Yamamori et al., 2013), we examined the expression of Rad51 and Ku70, which are required for the homology-directed and nonhomologous end-joining pathways of DSBR, respectively. Surprisingly, we found Rad51 and Ku70 protein levels to be repressed in SDS-LCLs compared to controls, both with and without exposure to IR. Collectively, these data support the hypothesis that, in addition to its role in ribosome biogenesis, SBDS is a stress response protein that plays an important role in regulating the ER stress response. In SDS-cells, where SBDS is lacking, activated ER stress represses DNA repair proteins rendering cells hypersensitive to IR and other stresses. This novel pathway to ER stress induction may contribute to the bone marrow failure and cancer predisposition seen in SDS patients. Disclosures No relevant conflicts of interest to declare.

Description: WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis) is a rare autosomal dominant immunodeficiency disorder that is caused by a gain in function in cysteine-X-cysteine chemokine receptor 4 (CXCR4) gene. Affected individuals have recurrent infections of the respiratory tract and soft tissues, and marked susceptibility to warts caused by human papilloma viruses (HPV). Patients have an increased risk of malignancy (lymphoma) and HPV-associated cancers. The optimal therapy for WHIM syndrome has not been defined and treatment has been with supportive care using intravenous immunoglobulin (IVIG) and granulocyte colony stimulating factor (G-CSF). New CXCR4 antagonist therapies are still in either phase I trials (plerixafor) or considered experimental (Chlacone-4). A successful umbilical cord blood transplant has been reported by Kriven et al. Here we describe a successful matched unrelated allogeneic stem cell transplant (SCT) in a girl with WHIM syndrome caused by a known mutation in the CXCR4 gene. This patient was diagnosed at birth with familial congenital neutropenia as findings were similar to those for the patient's mother and maternal grandmother. A complete blood count showed pancytopenia (WBC= 1.03 x103/µL, hemoglobin= 9.2 g/dL, platelet count= 74,000/µL, ANC= 70). Despite G-CSF and IVIG therapy, the patient had recurrent infections with sinusitis and pneumonia, along with progressive organomegaly with progressive pancytopenia. At the age of 4, the patient underwent matched unrelated SCT with a fully ablative regimen [busulfan 0.8 mg/kg/dose for 16 doses (days -9, -8, -7, and -6), cyclophosphamide 50 mg/kg/dose for 4 doses (days -5, -4, -3, and -2) and alemtuzumab 5 mg/day (weight-based dosage) for 3 doses (days -5, -4, and -3)]. Tacrolimus and mini methotrexate (on days +1, +3, +6 and +11) were given for GVHD prophylaxis. Neutrophil and platelet engraftment occurred on days + 21 and + 45, respectively. The patient is 100% donor chimeric. Post-transplant course has been complicated by grade II skin GVHD treated successfully with oral and topical steroids. The patient also developed adenovirus, BK virus and HHV-6 viral reactivations but without disease. The patient is now approximately 2 years post SCT with durable engraftment, normal humoral immune reconstitution and no chronic GVHD. Thus, stem cell transplant following myeloablative conditioning can be accomplished for patients with WHIM syndrome. Furthermore, transplantation performed at the patient's young age likely prevented complications of recurrent infections and warts. Disclosures Allen: NovImmune: Consultancy, Other: unpaid; Roche: Consultancy, Other: unpaid. Heslop:Cell Medica: Other: Licensing Agreement; Celgene: Other: Collaborative research agreement.

Description: Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by decreased or absent numbers of megakaryocytes and is not associated with congenital malformations. It is an autosomal recessive disorder with mutations in the thrombopoietin receptor c-MPL, presenting at birth with severe isolated thrombocytopenia. Given the increased risk of life threatening hemorrhage, close monitoring and supportive care with regular platelet transfusions is usually required. The severity of the MPL mutation may predict the clinical course of children with CAMT. Null mutations may rapidly progress to pancytopenia and aplastic anemia, while more modest functional loss of receptor function cause a transient increase in platelet count to 〉 50,000/µL during the first year of life with later progression to pancytopenia. Moreover, like other IBMFSs, CAMT has been referred to as a cancer predisposition syndrome. Allogeneic hematopoietic stem cell transplant (HSCT) offers the only curative option. We present our institutional experience of three patients with CAMT who underwent matched unrelated HSCT early in the course of the disease when their presenting problem was isolated thrombocytopenia without pancytopenia, marrow failure or clonal evolution. We have used a fully ablative regimen with busulfan 1 mg/kg/dose for 16 doses (days -9, -8, -7, and -6), cyclophosphamide 50 mg/kg/dose for 4 doses (days -5, -4, -3, and -2) and alemtuzumab 3 mg/day (weight-based dosage) for 4 doses (days -5, -4, -3, and -2). Cyclosporine and mini methotrexate (on days +1, +3, +6 and +11) were given for GVHD prophylaxis. The first two patients were siblings with persistent thrombocytopenia at birth, the first of whom had compound heterozygous mutations (c.256dupC and c.391+5 G〉C) in the MPL gene. Both parents were carriers and the second sibling was diagnosed prenatally with the same mutations. No other phenotypic abnormalities were noted and testing for Fanconi anemia was negative. The siblings were transplanted with matched unrelated donors at 12 months and 14 months respectively. Our third patient was diagnosed prenatally with germinal matrix hemorrhage and vetriculomegaly. He was noted to have thrombocytopenia after birth. He was treated initially for presumed neonatal alloimmune thrombocytopenia. Sequencing of the MPL gene revealed two compound heterozygous missense mutations (R257C and R102P). The patient was transplanted with a matched unrelated donor at the age of 11 months. All patients tolerated the transplant with minimal toxicity, durable engraftment, and no acute or chronic GVHD. The first two siblings are approximately 4 years and 2 years post HSCT and the third patient is past day 100. Previous reports of HSCT for CAMT that used matched unrelated donors recorded poor outcomes, with a high rate of graft failure. Although our current study is small in size, the results suggest that a HSCT following a fully ablative regimen containing alemtuzumab and performed early in the course of the disease may produce better outcomes, and will avoid the complications associated with marrow failure and clonal abnormalities. Disclosures Allen: Roche: Consultancy, Other: unpaid; NovImmune: Consultancy, Other: unpaid. Heslop:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Brenner:Celgene: Other: Collaborative Research Agreement; Cell Medica: Other: Licensing Agreement; Bluebird Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by pure red blood cell aplasia, variable congenital anomalies and increased risk of malignancy. Approximately 50-60% of cases are due to germline, heterozygous mutations in 1 of 13 genes that encode components of either the small (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26 and RPS29) or large (RPL5, RPL11, RPL15, RPL26, RPL31 and RPL35A) ribosomal subunits. The mutations may be either inherited or de novo. Consistent with DBA being a ribosomopathy, defects in ribosome assembly, altered ribosomal RNA processing and nucleolar stress are observed in cells of patients with DBA and ribosomal protein gene mutations. Mutations in GATA1, a hematopoietic transcription factor, have also been reported in rare X-linked recessive cases. Whole exome sequencing was independently carried out on two white/Hispanic probands with genetically uncharacterized DBA (BMF92 and NCI-62-1). Both presented with transfusion-dependent anemia during the first week of life. Bone marrow evaluations revealed marked erythroid hypoplasia characteristic of DBA. One of the probands (BMF92) experienced a spontaneous remission of his anemia toward the end of the first year of life, possibly related to corticosteroids administered for bronchiolitis. Subsequently, his erythrocyte adenosine deaminase(eADA) level was measured and elevated [10.5 units (mol/min/gm hg); reference range 0.42-3.5 units]. BMF92’s hypoplastic anemia later recurred and was found to be steroid refractory. NCI-62-1 is a male, now 12 years of age and red cell transfusion dependent. BMF92 and NCI-62-1 are of normal stature and have no documented congenital anomalies, although both have experienced chronic colitis. In both cases, family history for DBA was negative and parental eADA levels were within normal limits. Whole exome sequencing of peripheral blood DNA, validated by Sanger or Ion Torrent targeted sequencing, identified heterozygous variants of unknown significance in the ribosomal protein gene RPS20. Parental studies revealed both variants to be de novo. The variants were mutated at the same genomic position, but resulted in different amino acid substitutions in RPS20 [hg19 chr8 56985758, c.251A〉T, p.I84N (BMF92) and 56985758, c.251A〉C, p.I84S (NCI-62-1)]. Buccal swab analysis performed in BMF92 confirmed the presence of the variant. Both variants were novel, based on inspection of several databases, including dbSNP, 1000 Genomes, NHLBI Exome Sequencing Project, clinical WES data at the Whole Genome Laboratory at Baylor College of Medicine, Kaviar, Human Genome Mutation Database and ClinVar, totally well over 10,000 individuals as well as the COSMIC database, which reports somatic mutations in cancer. In silicoanalyses were consistent with the variants being damaging and disease causing (PolyPhen-2, SIFT and Mutation Taster), affecting highly evolutionarily conserved residues (GERP, PhyloP and Sitewise likelihood-ratio score) and decreasing the stability of the protein structure (MUpro and I-Mutant2.0). The above data strongly suggest that the RPS20 variants in these cases are disease causing. A recent report implicated a germline RPS20 truncating mutation in a four-generation pedigree with familial nonpolyposis colorectal carcinoma; however, the mutation carriers were reported to not manifest features of DBA (Nieminen et al., Gastroenterology 2014). This raises the likelihood of allele-specific effects. Functional assays of our probands’ mutations, including quantification of RPS20 protein and ribosomal RNA precursor steady state levels in patient versus control lymphoblastoid cell lines as well as analysis of the impact of mutant RPS20 protein expression on p53 and downstream targets are underway to determine their contribution to the DBA phenotype. Disclosures No relevant conflicts of interest to declare.