Inherited human Apollo deficiency causes severe bone marrow failure and developmental defects

Inherited bone marrow failure syndromes (IBMFS) represent a group of disorders typified by impaired production of one or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant Høyeraal-Hreidarsson (HH) syndrome are rare IBMFS characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. Here we identified biallelic variants in the gene encoding the 5'-to-3' DNA exonuclease Apollo/SNM1B in three unrelated patients presenting with a DC/HH phenotype consisting of early onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly and/or intrauterine growth retardation. All three patients carry a homozygous or compound heterozygous (in combination with a null-allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients' cells showed signs of telomere fragility that were however not associated with global reduction of telomere length. Unlike patients' cells, human Apollo KO HT1080-cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS combining clinical hallmarks of DC/HH with normal telomere length.

Genetic, parental and lifestyle factors influence telomere length

The average length of telomere repeats (TL) declines with age and is considered to be a marker of biological ageing. Here, we measured TL in six blood cell types from 1,046 individuals using the clinically validated Flow-FISH method. We identified remarkable cell-type-specific variations in TL. Host genetics, environmental, parental and intrinsic factors such as sex, parental age, and smoking are associated to variations in TL. By analysing the genome-wide methylation patterns, we identified that the association of maternal, but not paternal, age to TL is mediated by epigenetics. Coupling these measurements to single-cell RNA-sequencing data for 62 participants revealed differential gene expression in T-cells. Genes negatively associated with TL were enriched for pathways related to translation and nonsense-mediated decay. Altogether, this study addresses cell-type-specific differences in telomere biology and its relation to cell-type-specific gene expression and highlights how perinatal factors play a role in determining TL, on top of genetics and lifestyle.

Disease Progression and Clinical Outcomes in Telomere Biology Disorders

Dyskeratosis congenita related telomere biology disorders (DC/TBDs) are characterized by very short telomeres caused by germline pathogenic variants in telomere biology genes. Clinical presentations can affect all organs and inheritance patterns include autosomal dominant (AD), autosomal recessive (AR), X-linked (XLR) or de novo. This study examined the associations between mode of inheritance with phenotypes and long-term clinical outcomes. Two hundred thirty-one individuals with DC/TBDs [144 male, 86.6% known genotype, median age at diagnosis 19.4 years (0-71.6)], enrolled in the National Cancer Institute's Inherited Bone Marrow Failure Syndrome Study, underwent detailed clinical assessments and longitudinal follow-up [median follow-up 5.2 years (0-36.7)]. Patients were grouped by inheritance pattern, considering AD-nonTINF2, AR/XLR, and TINF2 variants separately. Severe bone marrow failure, severe liver disease and gastrointestinal telangiectasias were more prevalent in AR/XLR or TINF2 disease, whereas pulmonary fibrosis developed predominantly in adults with AD disease. After adjusting for age at DC/TBD diagnosis, we observed the highest cancer risk in AR/XLR individuals. At last follow-up, 42% of patients were deceased with a median overall survival of 52.8 years [95% confidence interval (CI) 45.5-57.6] and the hematopoietic cell or solid organ transplant-free median survival was 45.3 years (95% CI 37.4-52.1). Significantly better overall survival was present in AD versus AR/XLR/TINF2 disease (p<0.01), while patients with AR/XLR and TINF2 disease had similar survival probabilities. This long-term study of the clinical manifestations of DC/TBDs creates a foundation for incorporating the mode of inheritance into evidence-based clinical care guidelines, and risk stratification in patients with DC/TBDs. clinicaltrials.gov NCT00027274

Age-Related Phenotypes Linked to Aberrant Expression and Localization of a Telomeric Protein

Telomere attrition is associated with telomere biology disorders and age-related diseases. In telomere biology disorders, telomere uncapping induces a DNA damage response that evokes cell death or senescence. However, a causal mechanism for telomere attrition in age-related diseases remains elusive. Telomere capping and integrity are maintained by shelterin, a six-protein complex. Rap1 is the only shelterin member that is not required for telomere capping and is expressed at non-telomeric genomic and cytosolic regions. The objective of this study was to determine aberrant phenotypes attributed to non-telomeric Rap1. To test this, we generated a Rap1 mutant knock-in (KI) mouse model using CRISPR/Cas9 editing, in which Rap1 at telomeres is prevented, leaving only non-telomeric Rap1. Cell fractionation/western blotting of primary fibroblasts from Rap1 KI mice demonstrated decreased Rap1 expression and Rap1 re-localization off telomeres, with an altered cellular distribution. This same difference in Rap1 is also observed in human cells with telomere erosion, indicating that aberrant Rap1 in our model may recapitulate Rap1 in aging and human telomere biology disorders. Compared to wild-type control mice, Rap1 KI mice exhibited increased body weight, altered cytokine levels, behavioral deficits, and decreased lifespan. In conclusion, our results reveal a novel mechanism by which telomere shortening may contribute to age-related pathologies by disrupting Rap1 expression and cell localization.

Gain-of-Function Mutations in RPA1 Cause a Syndrome with Short Telomeres and Somatic Genetic Rescue

Human telomere biology disorders (TBD)/short telomere syndromes (STS) are heterogeneous disorders caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify three germline heterozygous missense variants in RPA1 gene in four unrelated probands presenting with short telomeres and varying clinical features of TBD/STS including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, or skin manifestations. All variants cluster to DNA binding domain A of RPA1 protein. RPA1 is a single-strand DNA-binding protein required for DNA replication and repair and involved in telomere maintenance. We showed that RPA1E240K and RPA1V227A proteins exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function while RPA1T270A has binding properties similar to wild type protein. To study the mutational effect in a cellular system, we used CRISPR/Cas9 to knock-in the RPA1E240K mutation into healthy inducible pluripotent stem cells. This resulted in severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in patient with RPA1E240K, we discovered somatic genetic rescue (SGR) in hematopoietic cells due to an acquired truncating cis RPA1 mutation or a uniparental isodisomy 17p with loss of mutant allele, coinciding with stabilized blood counts. Using single-cell sequencing, the two SGR events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD/STS.

Whole Exome Sequencing in Severe Aplastic Anemia Identifies Unrecognized Inherited Subset with Inferior Survival after Hematopoietic Cell Transplant

Background: It is important to identify inherited bone marrow failure syndromes (IBMFS) in patients with aplastic anemia in order to provide appropriate therapy. IBMFS are diagnosed through genetic or other diagnostic testing but can also go unrecognized, particularly in adults who may lack classic IBMFS features. This is particularly critical in light of increasing identification of individuals with variants of uncertain significance (VUS) and those with single pathogenic variants in an autosomal recessive (AR) gene or X-linked recessive (XLR) gene in females (SPVR) ("carriers") during evaluation. In this study of patients diagnosed with acquired severe aplastic anemia (SAA), we evaluated putatively causal variants in IBMFS genes to determine the frequency of patients with an unrecognized IBMFS or SPVR and assessed their association with outcomes after hematopoietic cell transplant (HCT). Methods: We used pre-HCT blood samples and clinical data from the Transplant Outcomes in Aplastic Anemia study (TOAA; a collaboration between the National Cancer Institute and the Center for International Blood and Marrow Transplant Research). Germline whole exome sequencing was performed on 732 patients with acquired SAA who received HCT between 1991-2015. A total of 104 IBMFS genes, (51 autosomal dominant (AD), 46 AR, 3 both AR and AD, 4 XLR) were evaluated for both single nucleotide (SNV) and copy number variants (CNV). All variants were curated using ACMG/AMP criteria, and a subset were validated by Sanger sequencing. Variants classified as VUS according to ACMG/AMP criteria and with a damaging score prediction in 3 of 5 in silico meta-predictors were categorized as deleterious VUS. Patients were divided into 3 groups based on known inheritance patterns of identified genes into those with 1) unrecognized IBMFS 2) SPVR or 3) neither (presumed acquired SAA). For telomere biology genes with AD/AR inheritance, we used telomeres <10 th percentile for age (by qPCR) to distinguish IBMFS from SPVR. For statistical analysis, we used the Kaplan-Meier estimator to calculate the probability of overall survival. The log-rank test was used to compare the survival distribution across patient categories. Cox proportional hazard models were used for multivariable analysis. Results: We identified 309 variants of them 156 were pathogenic or likely pathogenic (P/LP) (112 (71.8%) loss of function SNVs, and 10 CNVs), and 153 were deleterious VUS. Patients with deleterious VUS did not have different survival compared with those with no variants, and these were not considered for designating unrecognized IBMFS and SPVR cases Our analysis showed that 6.7% (N=49/732) of patients had variants consistent with an unrecognized IBMFS; 22 were AR (21 compound heterozygous, 1 homozygous), 26 AD and 1 XLR. Approximately half of those patients (22/49, 45%) had P/LP variants in hematopoiesis genes, and 31% in telomere biology genes (Figure 1A). We identified 79 patients with an SPVR, most in SBDS and FANCM. Patients with an unrecognized IBMFS had worse overall survival when compared with patients with presumed acquired SAA (log-rank p=0.0098) (Figure 1B). Multivariable analysis confirmed this association (HR=2.11, 95% confidence interval (CI)=1.38-3.22, p=0.001). The observed survival difference was not mitigated by lower conditioning regimen intensity (HR=2.1, p=0.01 in myeloablative condition (MAC), and HR=2.3, p=0.016, in reduced intensity regimens (RIC)). Patients with an SPVR had no post-HCT survival difference than presumed acquired SAA regardless of conditioning regimen (overall HR=0.96, p=0.85; MAC HR=1.4, p=0.27; RIC HR=0.3, p=0.1). Conclusions: A sizable subset of patients (6.7%) with reported immune mediated acquired SAA had unrecognized inherited disorder and 33% were adults at HCT. Unrecognized IBMFS was associated with statistically significant poorer survival after HCT. In contrast, post-HCT survival in patients with an SPVR ("carriers") was not different than those with acquired SAA. This work underscores the importance of identifying SAA patients with clinically meaningful underlying inherited disorders to enable the use therapeutic approaches to minimize regimen toxicity and late complications. It further highlights that identification of a single pathogenic variant in an autosomal recessive gene or X-linked gene in females and VUS are associated with post-HCT survival similar to acquired SAA. Figure 1 Figure 1. Disclosures Paczesny: Medical University of South Carolina: Patents & Royalties: inventor on the ST2 bispecific antibody patent application. Lee: Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; JANSSEN: Other; AstraZeneca: Research Funding; Takeda: Research Funding; Novartis: Other: clinical trials, Research Funding; Syndax: Research Funding; Pfizer: Research Funding; Incyte: Research Funding.

Unexpected High Frequency of Pathogenic Germline Variants in Older Adults with Primary Myelodysplastic Syndrome

Background: Germline predisposition is increasingly being recognised in myeloid neoplasms (MN) including primary myelodysplastic syndrome. An unequivocal diagnosis of germline predisposition carries actionable considerations for patient management including donor stem cell source for allogeneic transplantation, dose-reduction of conditioning regimes and screening for extra-hematological disease (such as pulmonary abnormalities in patients with telomere biology disorders). In addition, the identification of MDS predisposition syndromes can avoid misdiagnosis (for example, distinguishing idiopathic thrombocytopenic purpura from thrombocytopenia due to RUNX1 germline variant). However, the prevalence of pathogenic germline variants (PGVs) in unselected pMDS patients presenting at older age remains unknown. Aim: This study assesses frequency and type of pathogenic germline variants in MDS patients and compares with age matched healthy controls and patients with other cancers. Method: We analysed 68 known cancer predisposition genes in germline samples of 146 samples from myeloid neoplasms. Study included primary MDS (n=51) and MDS diagnosed in cancer survivors with (n=77) or without prior exposure to cytotoxic therapy (n=18). Using uniform American College of Medical Genetics and Genomics (ACMG) guidelines for annotating germline mutation, we also compared the frequency of pathogenic germline variants in the same genes with patients with single cancer and age-match healthy controls (>70 years). Results: Pathogenic germline variants (PGVs) were identified in 19% (28/146) patients compared to 4% and 3% patients with single cancer and age-matched controls respectively (P<0.0001) (Fig. 1A). Median age at diagnosis was similar between MN patients with or without PGVs [66 years (19-81) vs. 70 years (33-87); P=0.06]. PGVs were most frequent in DDX41 (n=7, 33%) followed by BRCA1 (n=2, 10%), GATA2 (n=2; 10%) and TP53 (n=2; 10%) (Fig.1B). We also identified pathogenic copy number variations (CNV) in 4 patients. The distribution of PGVs was also different, with DDX41 PGVs absent in single cancers and more prevalent in MN than age-matched controls (35% vs. 4%, P<0.001). The frequency of PGV was not significantly different between P-MN and T-MN/ MC-MN (17% versus 10%, P = 0.32 (Fig.1C). The frequency of PGV was 30%, 6%, 19%, 15% and 18% in patients ≤50, 51-59, 60-69, 70-79 and >80 years of age (Fig. 1D). Phenotypic features such as monocytopenia and mycobacterium infections (MonoMAC; SA460) and personal and family history of pulmonary fibrosis (SA918) were present in only two cases with PGVs. Family history of MDS/AML was present in only in four cases with PGVs, in which PGVs were found in typical myeloid malignancy genes (DDX41, GATA2). Importantly, some patients with family history of solid cancers carried PGVs in genes traditionally associated with solid cancers (e.g. MSH6, NF1, TP53 and BRCA1). SA927 had a PGV in MSH6 and multiple first-degree relatives with solid cancers including colon, renal and brain cancers. Moreover, 41% of adults with hematological disorders and a personal and/or family history of interstitial lung disease had PGVs in telomere biology disorder genes. Hence, family history should not be restricted to hematological disorders, but also solid cancers and non-malignant phenotypes (e.g. hepatic and pulmonary fibrosis). The frequency of PGVs was not different in patients with and without family history of cancer (23% vs. 13%, P=0.32). Conclusion: The frequency of PGVs is significantly high in MN compared to age matched healthy control and other cancer patients. Our observation of a high frequency of PGVs in the older MDS population warrants standardization of germline testing at diagnosis to guide optimal management of patients and their families. Figure 1 Figure 1. Disclosures Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

U2AF1 and Other Splicing Factor Gene Mutations in Telomere Biology Disorders Are Associated with Hematologic Neoplasia and Worse Overall Survival

Introduction: Telomere Biology Disorders (TBD) are due to germline variants in telomere maintenance and repair genes. Clinical manifestations include bone marrow failure (BMF), liver and lung fibrosis, and risk of cancer, especially myeloid neoplasia. We characterized clinical phenotype, pathology, and clonal landscape of patients with TBD and myelodysplastic syndrome (MDS) or acute leukemia (AL). We assessed if somatic variants in myeloid cancer genes are associated with MDS/AL or overall survival (OS). Methods: Patients (n=109) from the National Institutes of Health and University of São Paulo (NIH/USP) (n=91) or MD Anderson (MDA) (n=18) met TBD clinical criteria or had a pathogenic/likely pathogenic germline variant in a telomere maintenance/repair gene. All patients gave informed consent. Clinical characteristics and details of hematologic malignancy were collected. MDS was defined using WHO 2016 criteria. In the NIH/USP cohort, somatic variants in myeloid cancer genes were assessed in peripheral blood using error-corrected DNA sequencing (ECS; minimum variant allele frequency [VAF] of 0.5%) and in the MDA cohort patients were screened by targeted amplicon-based next-generation sequencing (minimum VAF of 2%). Serial samples were available in 31 (41%) patients. Results: Eighteen out of 109 (17%) patients from both the NIH/USP and MDA cohorts had a diagnosis of MDS/AL, developing in patients with TERC (n=8/31; 25%), TERT (n=8/48; 16%), RTEL1 (n=1/5; 20%), and DKC1 (n=1/2; 50%). Karyotypes were normal diploid (n=6), chromosome 1 abnormality (n=5), trisomy 8 (n=2), deletion 20q (n=1), complex (n=2), monosomy 7 (n=1) and deletion 7q (n=1). Six patients had >5% blasts. Four patients had concurrent liver fibrosis and 9 had pulmonary fibrosis. Somatic mutation data at time of MDS/AL was available for 16/18 patients; splicing factor genes (n=14) were most frequent, seen in 11 (69%) patients, with U2AF1 predominating (n=7; 44% of patients) (Figure 1A). More than 1 mutation was present in 12/16 (75%) of patients. One patient developed AML 6 years after MDS diagnosis . U2AF1 variants were also present in 8 patients without MDS/AL; 5 had no sequential samples and 3 had stable mutations over 2 (n=2) and 4 (n=1) years. Recurrent variants in U2AF1 were present at p.S34 (n=12) and p.Q157 (n=2). Median U2AF1 variant allele fraction (measured in NIH/USP cohort only) was 23% in patients with MDS/AL compared to 7% in those without. Variant association analysis was confined to NIH/USP cohort due to DNA sequencing assay differences; data were available in 75 patients. Patients were divided into groups defined by specific variants at any time of sampling; "Clonal-hematopoiesis of indeterminate potential / aplastic anemia (CHIP/AA) associated" (DNMT3A, TET2, ASXLI, or BCOR/L1), "MDS-associated" (containing splicing factor genes, RUNX1, SETBP1, ETV1, KRAS, STAG2, GATA2/1, TP53), "PPM1D" (containing PPM1D), and a subgroup, "Splicing factors" (U2AF1, ZRSR2, SF3B1, SRSF2). Patients with MDS-associated variants (Figure 1A; p=0.02), particularly splicing factors (Figure 1B; p=0.007) more likely had or later developed MDS/AL, compared to patients without somatic variants or in other variant groups. MDS/AL was not associated with PPM1D or CHIP/AA-associated variants. OS from time of first detected somatic variant was lower in the MDS-associated variant group (Figure 1C p=0.013) compared to those without variants at time of first assessment; this was not the case for CHIP/AA-associated variants or PPM1D. Patients with somatic variants (median ages 36, 41, and 42 for PPM1D, CHIP/AA-associated, and MDS-associated respectively) were significantly older than patients without variants (median age 24) though ages were similar within variant groups. Conclusion: Splicing factor gene variants predominate the clonal landscape of TBD-associated hematologic malignancy; U2AF1 p.S34 is recurrent and can occur in the absence of neoplasia. Splicing factor gene variants such as U2AF1 in the peripheral blood may be potential biomarkers for hematologic malignancy in TBD patients. PPM1D, previously associated with therapy-related MDS, is commonly mutated in TBD but does not associate with MDS/AL. OS was decreased in patients with MDS-associated variants but not with PPM1D or CHIP/AA-associated variants. Further study of U2AF1 variants in TBD patients may give insight to the underlying drivers of MDS/AL. Figure 1 Figure 1. Disclosures DiNardo: Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Forma: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Takeda: Honoraria; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Calado: Novartis Brasil: Honoraria; Instituto Butantan: Consultancy; AA&MDS International Foundation: Research Funding; Alexion Brasil: Consultancy; Agios: Membership on an entity's Board of Directors or advisory committees; Team Telomere, Inc.: Membership on an entity's Board of Directors or advisory committees. Young: Novartis: Research Funding.

Clonal Hematopoiesis in Telomere Biology Disorders Associates with the Underlying Germline Defect and Somatic Mutations in POT1, PPM1D, and TERT promoter

Introduction: Telomere biology (TBD) disorders are caused by pathogenic germline variants in genes related to telomere maintenance. In TBD, clonal hematopoiesis (CH) has been hypothesized to compensate for restricted cell fitness and to lead to development of myelodysplastic syndromes and acute myeloid leukemia (MDS/AML). We sought to characterize the clonal landscape and dynamics by deep sequencing of a large cohort of TBD patients with a broad spectrum of phenotypes and ages. Methods: We screened 120 TBD patients (median age=29) from the National Institutes of Health and the University of Sao Paulo for somatic mutations in genes related to myeloid malignancies and telomere diseases using an error-correcting DNA sequencing panel (minimum allele frequency [VAF] of 0.5%). Patients had either a pathogenic germline variant in telomere-related genes or short telomeres in blood and a strong clinical suspicion for TBD. Relatives were included if they harbored the proband's germline mutation. Single-cell DNA sequencing was performed in marrow samples from two TBD patients with MDS (TBD-MDS) to elucidate clonal trajectories Results: Fifty-eight TBD patients (48%) had somatic mutations in peripheral blood (median age and range, 42 years; 9-57), most frequently in PPM1D (all exon 6 truncated; n=18) , TERTp (-57, -124, and -146; n=14), POT1 (n=12), U2AF1 (n=12), and other MDS-associated genes. Clinically, these patients had dyskeratosis congenita (DC; n=12/27), aplastic anemia (AA; n=11/27), isolated cytopenias (n=7/10), MDS/AML (n=7/8), pulmonary or liver fibrosis (n=4/8), and multi-organ disease (n=19/26). In this series, no relatives had somatic mutations (n=14). CH frequency increased with age and was significantly more frequently observed than in healthy controls, regardless of age (p<0.001). POT1, PPM1D, and TERTp clones size was lower than the size of MDS-associated clones (VAF of 1% vs 8%). These mutations often co-occurred, except for POT1 and TERTp mutation. Patients' clonal profiles correlated with the underlying germline defect. Somatic P OT1 mutations strongly associated with TINF2 germline variants, and consequently DC: 5/9 TINF2 patients had one (n=2) or >2 POT1 clones (n=3). In contrast, both TERTp and PPM1D clones were mostly detected in TERT/TERC patients with multi-organ disease, especially pulmonary fibrosis and marrow failure. No telomere elongation or improved blood counts were seen in serial samples. TINF2 patients with somatically mutated POT1 clones were older despite their DC diagnosis (median age=19 vs 5 years in POT1 mutated and wild type, respectively). A single patient with a germline TINF2 R282C and somatically mutated POT1 clone at VAF=29%, which was stable for 5 years, had MAA. The median ages (range) of TERT/TERC patients with TERTp and PPM1D mutations were 41 (25-64) and 43 (12-72), respectively, whereas TERT/TERC patients without TERTp and PPM1D mutations were at a median age of 27 (8-58). Most clones were stable regardless of clinical phenotype, even after danazol treatment. PPM1D clones were stable for 2-9 years of follow-up. TERTp and POT1 clones' size decreased while on androgens but consistently increased after the drug was discontinued. In single-cell DNA analysis of two TBD-MDS patients, the U2AF1 S34F and Q157R were driver mutations and occurred with mutations in RUNX1, ETV6, ASXL1; these clones were stable for 3-6 years. In the first case, the U2AF1 clone subsequently acquired a RUNX1 mutation; this clone was coincident with an independent clone containing PPM1D and POT1 mutations. In the second patient, a U2AF1 clone acquired successive mutations in SETBP1 and AXSL1; a second clone with U2AF1 and additional mutations in GATA2 and KRAS arose at evolution to AML. Conclusion: In TBD, the somatic landscape differed from age-related CH, with recurrent TERTp, POT1, and truncated PPM1D mutations. Mutations' frequency increased with age but was related to the underlying germline mutation. It is uncertain whether clonal selection is a probabilistic consequence of older age or the cause of mild phenotypes and prolonged lifespan. Despite the association of POT1 and PPM1D with malignancies, no patients in our cohort had POT1-related cancers or had received chemotherapy. POT1 and PPM1D like TERTp mutations may arise to compensate for cell fitness. Clinically, this distinct clonal landscape, not found in immune BMF, could serve as a molecular marker of underlying TBD. Disclosures Calado: Instituto Butantan: Consultancy; AA&MDS International Foundation: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis Brasil: Honoraria; Alexion Brasil: Consultancy; Team Telomere, Inc.: Membership on an entity's Board of Directors or advisory committees. Young: Novartis: Research Funding.

Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins

DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon we have investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on our results and on homology-based three-dimensional models for Ver and Moi, we conclude that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. We propose that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. We also provide here the first evidence on Moi DNA binding.