ALBERT

Description: Myelodysplastic syndromes (MDS) following chemo- and/or radiotherapy for primary malignancy in childhood are clinically heterogeneous and generally resistant to therapy. Current knowledge about the clinical course and the underlying genetic background is imprecise with the exception of structural complex karyotype indicating a particular dismal prognosis. Here, we employed a next generation sequencing (NGS) of 28 genes previously established to assess the mutational landscape in primary pediatric MDS. Using Ampliseq PCR enrichment and Miseq NGS we deep sequenced DNA from bone marrow of 80 patients (pts) with therapy-related MDS (t-MDS) included in the registry of the European Working Group of MDS in Childhood (EWOG-MDS). The 41 males and 39 females had been diagnosed of t-MDS at a median age of 13 years (yrs, range 1.2-22) following treatment of hematopoietic malignancy (HM, 44), solid tumor (ST, 22) or brain tumor (BT, 14). The interval from primary malignancy to t-MDS was 3.2 yrs (0.5-18.5). Morphology was compatible with refractory anemia with excess of blast (RAEB, 34), RAEB in transformation (RAEB-t, 9), MDS related acute myeloid leukemia (MDR-AML, 4), chronic myelomonocytic leukemia (CMML, 14) or MDS without increase of blasts (19). Cytogenetic aberrations were seen in 80% of the patients, most frequently monosomy 7 (29%) and a structural complex karyotype (22%) detected in all morphological entities and subgroups of primary malignancy. Mutations were noted in 50% of pts with 1, 2, 3 or 4 genes affected in 33%, 10%, 6% and 1% respectively. Recurrent mutations were identified in RUNX1 (13.8%), TP53 (12.5%), CBL (8.8%), ASXL1 (7.5%), PTPN11 (7.5%), SETBP1 (5.0%), NRAS (5.0%), KRAS (3.8%), BCORL (2.5%), CSF3R (2.5%), HOXA9 (1.3%), EZH2 (1.3%), PTEN (1.3%), JAK3 (1.3%), STAG2 (1.3%), RAD21(1.3%). No mutations were detected in GATA2, CEBPA, GATA1, BCOR, FLT3, JAK2, cKIT, MPL, CTCF, VPS45, CALR, SH2B3. TP53 mutations arose as an isolated genetic event in the majority of cases (8/10) unlike RUNX1 mutations frequently occurring in combination with other genetic lesions (9/11). According to primary malignancy TP53 mutations were detected in all subgroups (14% HM, 63% ST, 14% BT), whereas RUNX1 mutations were absent in the group with prior solid tumor. Mutations in TP53 were strongly correlated with a structural complex karyotype (9/10). With the exception of MDS without increase of blasts characterized by the absence of RAS pathway alterations, the mutational landscape did not segregate with the morphological subtype. With a median follow-up of 5.3 yrs (0.1-13.7) for pts alive and 68/80 pts treated with hematopoietic stem cell transplantation (HSCT), the probability of overall survival (OS) at 5 yrs was 0.44. OS was worse for patients with more advanced disease (RAEB-t/MDR-AML 0.12 vs CMML 0.29 vs RAEB 0.49 vs MDS without increase of blasts 0.61, p=0.07) and t-MDS following solid tumor (ST 0.30 vs HM 0.49 vs BT 0.46, p=0.09). As expected the detection of a structural complex identified a group with a dismal outcome (OS 0.09 vs 0.37 with normal karyotype vs 0.55 with monosomy 7/ 7q- vs 0.62 with other cytogenetic aberrations, p

Description: Dyskeratosis congenita (DC) is a rare telomere disease with pleiotropic manifestations and bone marrow failure (BMF) as a major cause of mortality. The genes involved in DC pathogenesis play a role in telomere maintenance but their function in other in biological systems has not been well characterized. Compound heterozygous missense mutations in TCAB1(WRAP53) were reported so far in two pedigrees with classic DC (Zhong et al, 2011). TCAB1 functions as a scaffold protein recruiting TERC to the sub-organelle Cajal bodies, thus contributing to the assembly of telomerase enzyme; among its other functions it regulates DNA double-strand break repair. Here we functionally characterize novel TCAB1 mutations and describe transcriptome and proteome profiles in patient-derived lymphoblastoid cell lines (LCL). Among a cohort of 50 DC patients we identified three pedigrees with TCAB1 mutations, BMF and telomeres below first percentile. Index 1 carried compound heterozygous mutations R155X/Y345C also present in the affected brother. Both siblings suffered from transfusion-dependent pancytopenia manifesting in the childhood and typical dyskeratotic features. Index 2 harbored a single mutation Q7TfsX27 and presented with hypocellular BMF. Although non-hematologic symptoms were initially absent, the patient suffered from mild restrictive lung disease after stem cell transplantation. Index 3 carried biallelic mutations Y345C/G435R. He was diagnosed with microcephaly, cerebellar hypoplasia, early onset BMF with immunodeficiency, consistent with Hoyeraal-Hreidarsson syndrome (HHS). To gain insight into the functional consequences of novel mutations identified, we first assessed RNA, protein levels and localization in patient-derived LCLs and HeLa cells stably expressing wildtype (WT) and mutant TCAB1. The truncating mutations R155X and Q7TfsX27 were unable to translate to protein with abrogative localization in Cajal bodies, while the R155X mutation affected RNA stability through nonsense-mediated decay. The Y345C mutant was translated, but it was severely diminished in the nucleus and demonstrated defective localization in the Cajal bodies. The decline of TCAB1 signal in Cajal bodies was also observed in patients 1&3 with biallelic mutations. Next, to describe the effect of mutations on cellular processes we performed RNA-sequencing (RNAseq) and mass spectrometric-based quantitative proteomics using SILAC on LCLs from patients/carriers and WT controls. The principal component analysis (PCA) of RNAseq data clusters the transcriptomes of patients with missense mutations (Y345C/G435R, Y345C) and truncating mutations (R155X, Q7TfsX27) into separate groups, while index 1 (R155X/Y345C) demonstrated a very distinct transcriptomic profile (Fig 1A). The telomere maintenance processes were highly downregulated for index 1 and clinically healthy carrier parents (Fig 1B). This might suggest that heterozygous mutations also affect telomere biology; however the compensatory effect from the WT allele allows maintaining normal homeostasis. Based on disease severity and established pathogenic effect of mutations we then focused on index patients 1 and 3 carrying biallelic mutations. The differential gene expression analysis suggested RPSA, LILRB1 (downregulated) and SH3BP5, TSPYL5 (upregulated) as top candidates. Strikingly, the identification of downregulated RPSA points at dysregulated ribosomal function as a possible novel mechanism in TCAB1-mutated cells. Finally, positive correlation was observed for differential gene regulation on transcriptome and protein level. A gene set variation analysis indicated down-regulation of telomere maintenance, protein processing and ubiquitination. Surprisingly the members of the telomerase holoenzyme complex were enriched on the protein level however they were detected transcriptionally down-regulated in patients (Fig 1C). This might hint at defective protein transport or slower protein turnover due to decreased ubiquitination. In summary, we expand the clinical spectrum of TCAB1-associated DC ranging from BMF without mucocutaneous symptoms to severe HHS. We provide additional insights into the underlying cellular effects of TCAB1 mutations. The preliminary insight into the gene networks affected by TCAB1 mutations allows for first comprehension into the pleiotropic effects observed in patients. Disclosures No relevant conflicts of interest to declare.

Description: Dyskeratosis congenita and related telomere biology disorders (DC/TBDs) are multisystem illnesses caused by germline pathogenic variants (PV) in telomere biology genes resulting in very short telomeres. The inheritance varies and includes autosomal dominant (AD), autosomal recessive (AR) and X-linked (XLR) disease. Young patients may present with bone marrow failure (BMF) and the mucocutaneous triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia, whereas adults may be identified with isolated BMF, pulmonary fibrosis (PF), or liver disease. In addition to BMF, DC/TBDs are associated with high risk of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), head and neck squamous cell cancer (HNSCC) and many other medical problems. Hematopoietic cell transplant (HCT), lung, and/or liver transplantation may be required. A limited number of genotype-phenotype studies suggest the mode of inheritance to be associated with phenotypes. We studied 229 individuals (144 males, 85 females) with DC/TBDs enrolled in an IRB-approved longitudinal cohort study of inherited BMF syndromes (NCT00027274) between 01/2002 and 05/2019. Questionnaires and medical records were reviewed and a subset of 93 patients (40.6%) was systematically evaluated at the NIH Clinical Center (CC) during a 5-day visit. The median age at diagnosis for the entire cohort was 23.4 (0-82.2) years and median follow-up time was 4.2 (0-36.2) years. The PV was identified in 180 (78.6%) patients: 98 had AD disease (TERT, TERC, RTEL1, ACD, or PARN), 58 had XLR or AR (TERT, RTEL1, ACD, WRAP53, PARN, or DKC1) and 24 TINF2 (AD or de novo). In the entire cohort of 229 patients, 69 (30.1%) required HCT for BMF [median age 14.9 (0.9-63.1) years] and 58% of these had AR/XLR or TINF2-associated disease. Eight patients received lung transplant for PF [median age 51.4 (13.1-62.4) years]. Liver transplant was required in 5 patients [median age 25.8 (9.9-56.7) years]. Twenty-seven non-HCT patients developed cancer [median age of 42.1 (18.2-67.5) years]. In 5 patients, cancer occurred after HCT [median time since HCT=4.6 (1.8-14.6) years] and 3 developed post-HCT lymphoproliferative disease. Ninety-three patients were systematically phenotyped at the CC; 52 (55.9%) had 〉=1 triad feature and 33 (35.5%) had severe BMF. Three non-HCT patients had a history of cancer. PF was present in 6 patients and 3 had severe liver disease (portal hypertension and/or liver cirrhosis/fibrosis). Esophageal strictures were present in 7 and 5 had hip avascular necrosis (AVN, non-HCT). Median follow-up time after CC visit was 7.1 (0-17.1) years with median age at last follow-up of 29.6 (2.2-79.5) years . During follow-up, 9 patients progressed to severe BMF, and 8 developed cancer (4 post-HCT). Ten developed PF (5 post-HCT), and 9 advanced to pulmonary arteriovenous malformations (8 post-HCT). Eight patients progressed to severe liver disease (7 post-HCT). Gastrointestinal telangiectasias were seen in 6 individuals. Four patients developed esophageal stricture and 10 AVN (7 after radiotherapy and/or HCT). At last follow up of the entire cohort, 92 (40.2%) of 229 patients were deceased. The main causes of death were lung disease (28.3%), treatment-related complications (19.6%), cancer (16.3%), or BMF (14.1%). The median overall survival was 54.2 years [95% confidence interval (CI) 47.3-59.8] and HCT-free median survival was 46.9 years (95% CI 37.9-54.2). Overall survival was significantly better in patients with AD versus XLR/AR or TINF2 disease (p

Description: Diamond-Blackfan anemia (DBA) is a prototypic ribosomopathy and remains the most common cause of congenital pure red cell aplasia (PRCA). In 2/3 of patients, ribosomal protein haploinsufficiency is disease-causing, while in remaining 1/3 the genetic etiology is unknown. Recently, deficiency of ADA2 (DADA2) due to biallelic CECR1 -mutations was reported in patients with systemic autoinflammatory disease presenting with early onset vasculopathy, strokes, antibody deficiency, and in some cases variable cytopenias. Based on the clinical findings in an ADA2-deficient patient with PRCA resembling DBA, we aimed to define the prevalence and clinical picture of DADA2 within DBA patient cohorts. Patients enrolled in the national observational DBA registry in Germany were evaluated for the presence of mutations in CECR1 gene; additional nonconsecutive patients from the French and Turkish registries within the European DBA (EuroDBA) consortium were part of this study. Functional studies included profiling of polysomes and pre-rRNAs in patient-derived EBV-cell lines, CECR1 RT-PCR, measurements of autophagy and apoptosis, and analysis of erythropoiesis in zebrafish embryos. Systematic mutational and copy number analysis had identified typical ribosomal haploinsufficiency in 169/242 patients (70%). Out of 73 remaining patients, full CECR1 -sequencing was accomplished in 68 cases, of which 4 (6%) carried biallelic CECR1 -mutations. Additional 3 patients with biallelic CECR1 -mutations and DBA phenotype were referred from Germany (the index PRCA case), France and Turkey. In contrast to typical autoinflammatory DADA2 (caused by missense biallelic CECR1 -mutations) all patients studied here had at least one CECR1 -allele affected by truncating/stop-gain/deletion mutation leading to mRNA degradation in patient cells. Low or missing ADA2 enzyme activity in plasma confirmed DADA2, while erythrocyte ADA (eADA) levels and MCV were normal. Transfusion-dependent hypoproliferative anemia developed at a median age of 5 weeks (birth-14 years), while hypogammaglobulinemia developed in all cases either initially or during disease course. Notably, a transient hematologic response to steroids was achieved in 5/7 patients, but no improvement was observed in 2 patients treated with TNF-inhibitor; all patients at one point became heavily transfusion-dependent. Systemic vasculitis or cerebral complications were not observed in our cohort. At the last follow-up, 6/7 patients were alive; 3 had successfully undergone hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning and 1 patient had died due to septic shock. Next, we addressed the question if ribosome biogenesis is affected in ADA2-deficient patient cells. Using pre-rRNA maturation assays, polysome profiling and Western blots we established that ribosome biogenesis is normal in DADA2-related PRCA and there is no increase of TP53 stabilization over basal levels in patient LCLs. Analysis of CECR1 -morpholino zebrafish embryos revealed early anemia with lethal phenotype. Although there was no evidence for extrinsic (e.g. immune-mediated) pathomechanisms in our patients, it remains to be answered if CECR1 loss directly affects erythroid development. Finally, the association between elevated levels of eADA (=ADA1) specific to classical DBA and decreased ADA2 enzyme levels in DADA2-related PRCA remains obscure. In summary, DADA2 can phenotypically mimic DBA and thus extends the spectrum of congenital PRCA. Ribosome synthesis seems not to be affected by CECR1 mutations. DADA2 should be considered in patients with DBA-like phenotype but with normal eADA/MCV and hypogammaglobulinemia, allowing for early stratification aimed at HSCT in affected individuals. Disclosures Grosse: Addmedica: Membership on an entity's Board of Directors or advisory committees.