No abstract

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome caused by mutations in 7 genes involved in telomere biology, with approximately 50% of cases remaining genetically uncharacterized. We report a patient with classic DC carrying a compound heterozygous mutation in the CTC1 (conserved telomere maintenance component 1) gene, which has recently implicated in the pleiotropic syndrome Coats plus. This report confirms a molecular link between DC and Coats plus and expands the genotype-phenotype complexity observed in telomere-related genetic disorders.

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases.

Key Points PS can be overlooked in the differential diagnosis of children with severe congenital anemia. mtDNA deletion testing should be included in the genetic evaluation of patients with congenital anemia of unclear etiology.

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, nextgeneration sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.

Pearson marrow pancreas syndrome (PS) is a congenital multisystem disorder characterized by sideroblastic anemia, pancreatic insufficiency, metabolic acidosis, and other defects, and is caused by mitochondrial DNA (mtDNA) deletions. Diamond Blackfan anemia (DBA) is a congenital hypoproliferative anemia with associated physical malformations, and in which mutations in ribosomal protein (RP) genes and GATA1 have been implicated. The clinical presentation of both of these bone marrow failure (BMF) syndromes shares several features including early onset of severe anemia, sporadic genetic inheritance, variable penetrance and manifestations, and episodes of spontaneous hematologic improvement. PS is less frequently occurring than DBA, with estimated incidences of < 1/1,000,000 versus 1/100,000 respectively, and therefore less often encountered by hematologists. We hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. To test this hypothesis, we retrospectively evaluated DNA samples from a cohort of patients that were submitted to a research study for DBA genetic testing. The study cohort consists of clinical samples and/or data from 362 patients, with a primary inclusion criterion of known or suspected congenital anemia. Prior genetic studies from this cohort have yielded the novel identification or confirmation of mutations and deletions in several genes implicated in DBA (e.g. RP genes, GATA1), which are to date identifiable in 175/362 samples (48%), a proportion consistent with that found in independent DBA registries. We screened peripheral blood DNA samples available from 173 genetically uncharacterized patients using a long PCR strategy, and found that 8 samples (4.6%) contained large mtDNA deletions. Deletion mapping and Southern blot analysis on DNA from these 8 patients confirmed the presence of a single deletion event within each patient, ranging in size from 2.3 - 7.0 kb of the 16.6 kb mitochondrial genome, existing as monomer or multimer mtDNA species, and in a proportion ranging from 55-80% of total mtDNA, all of which are consistent with the molecular diagnosis of PS. Follow-up with referring providers in the 1 month to 8 year time span since sample submission revealed that 2 of the 8 patients (25%) were subsequently diagnosed with PS. Of the remaining 6 undiagnosed patients, 2 had died from complications of bone marrow transplantation, performed for worsening cytopenias and concern for myelodysplasia; one patient died from bacterial sepsis; and 3 were alive with the provisional diagnosis of DBA. One of the 3 patients had become transfusion-independent. Review of bone marrow examinations revealed that the pathological hallmarks of ringed sideroblasts and/or vacuolization of precursors described in PS were inconsistently present or reported in the diagnostic evaluation. We conclude that PS is frequently overlooked in the diagnostic evaluation of children with congenital anemia. Establishing the diagnosis of PS, as distinct from DBA and other BMF disorders, holds important implications for patient management and family counseling. mtDNA deletion testing should be performed in the initial genetic evaluation of all patients with congenital anemia. Disclosures: Szczepanski: Octapharma AG: Investigator Other.

Private germline mutations affecting hematopoiesis can cause progressive myelodysplasia and thus constitute pre-leukemic states. These can remain undetected, progressively transform and reveal themselves as infant leukemias, which can be linked to translocations involving the mixed lineage-leukemia (MLL) gene. Septin proteins play key roles in mammalian cell division and cytokinesis and are found as fusion partners of MLL in infant and early childhood acute myeloid leukemia (AML). We identified and describe a human germline disorder of septins in a newborn with myelodysplasia who required early hematopoietic stem cell transplant (HSCT) to prevent progressive disease. Materials & Methods: A Caucasian newborn male with no birth defects/malformations or suspicious family history presented with severe progressive neutropenia and was found to have bone marrow (BM) dysplasia with tetraploidy of myeloid progenitors. The patient developed unfavorable clonal aberrations (trisomy 7,8,9) and increased tetraploidy. Due to his progressive cytopenias and concern about risk of leukemic transformation, he underwent an allogeneic busulfan-cyclophosphamide/ATG conditioned DQ-mismatched unrelated HSCT at age 1 yo. He is currently 8 years post-HSCT with normal trilineage hematopoiesis (full donor chimerism), no graft versus host disease or any other non-hematological phenotype. To investigate the genetic etiology of this unique phenotype, we performed family trio germline exome/whole genome next-generation sequencing (NGS), and somatic pre-HSCT BM NGS for the index case. An established algorithm filtered for significant candidates following a de novo germline model. Immunohistochemical (IHC) staining of the pre- and post-HSCT BM biopsies for the candidate protein was performed. To validate the germline origin of the candidate mutation, we generated patient and control skin fibroblasts and induced pluripotent stem cells (iPSCs) that underwent fidelity testing by murine injection teratoma assays and 16-marker immunofluorescence (IF) staining. We then studied hematopoietic progenitor cells (HPCs) derived from iPSC-embryoid bodies (EB) in methylcellulose assays. To further determine the pathogenic nature of the mutation, we generated CRISPR/Cas9 knock-out of the human erythroleukemic cell line (TF-1) and studies these cells by cytomorphology, DNA and cell cycle assays. In-silico protein analysis of the candidate mutation and its effects on septin filament formation was performed. Results: Family trio and disease-tissue NGS identified a novel, germline C-terminal mutation in SEPT6, which was acquired de novo in the patient, and was not found in any database of common polymorphisms. IHC of pre-HSCT patient BM showed reduced Septin-6 staining in megakaryocyte and granulocyte precursors compared to post-HSCT and controls. Patient-derived iPSCs carried the mutation, were cytogenetically normal and bona-fide pluripotent by IF and teratoma assays. EB-derived HPCs from these cells recapitulated the patient's phenotype as they differentially failed to produce granulocyte vs erythroid colonies (fold-reductions CFU-M:8, CFU-G:36, CFU-GM:46, BFU-E:6, see Figure). Despite multiple approaches, SEPT6 CRISPR/Cas9 knock-out/in of the patient's mutation was not tolerated in iPSCs and human myeloid (granulo-/myelocytic) cell lines (HL-60, Molm-13, K562), and only tolerated in erythroid TF-1 cells. Analysis of SEPT6 knock-out TF-1 single-clone lines revealed a propensity to poly-nuclearity/lobation, as observed in the patient's BM. SEPT6 knock-in of C-terminal mutations caused cell death, consistent with existing literature. In silico protein analysis (incl. previously published crystallographic data) suggests that the mutation a) most likely modulates the key role of the coiled-coil SEPT6 domain in septin filament stabilization/bundling/bending, and thus deleteriously impacts cytokinesis, and b) perturbs the equilibrium of splice variants, possibly conferring tissue specificity. Conclusions: Mutation of the C-terminus of human SEPT6 causes aberrant cytokinesis in HPCs leading to a severe congenital neutropenia with tetraploidy and progressive myelodysplasia and cytogenetic aberrations. This report implicates a human germline disorder of SEPT6, and further investigations are required to elucidate the role septins in normal and disordered myelopoiesis. Figure. Figure. Disclosures Williams: Bluebird Bio: Research Funding.