Dyskeratosis congenita and the DNA damage response

Dyskeratosis congenita (DC) is a heterogeneous bone marrow failure disorder with known mutations in components of telomerase and telomere shelterin. Recent work in a mouse model with a dyskerin mutation has implicated an increased DNA damage response as part of the cellular pathology, while mouse models with Terc and Tert mutations displayed a normal response. To clarify how these contradictory results might apply to DC pathology in humans, we studied the cellular phenotype in primary cells from DC patients of several genetic subtypes, focussing on T lymphocytes to remain close to the haematopoietic system. We observed novel cell cycle abnormalities in conjunction with impaired growth and an increase in apoptosis. Using flow cytometry and confocal microscopy we examined induction of the DNA damage proteins γ-H2AX and 53BP1 and the cell cycle protein TP53 (p53). We found an increase in damage foci at telomeres in lymphocytes and an increase in the basal level of DNA damage in fibroblasts, but crucially no increased response to DNA damaging agents in either cell type. As the response to induced DNA damage was normal and levels of global DNA damage were inconsistent between cell types, DNA damage may contribute differently to the pathology in different tissues

Mutations in C16orf57 and normal-length telomeres unify a subset of patients with dyskeratosis congenita, poikiloderma with neutropenia and Rothmund–Thomson syndrome

Dyskeratosis congenita (DC) is an inherited poikiloderma which in addition to the skin abnormalities is typically associated with nail dystrophy, leucoplakia, bone marrow failure, cancer predisposition and other features. Approximately 50% of DC patients remain genetically uncharacterized. All the DC genes identified to date are important in telomere maintenance. To determine the genetic basis of the remaining cases of DC, we undertook linkage analysis in 20 families and identified a common candidate gene region on chromosome 16 in a subset of these. This region included the C16orf57 gene recently identified to be mutated in poikiloderma with neutropenia (PN), an inherited poikiloderma displaying significant clinical overlap with DC. Analysis of the C16orf57 gene in our uncharacterized DC patients revealed homozygous mutations in 6 of 132 families. In addition, three of six families previously classified as Rothmund–Thomson syndrome (RTS—a poikiloderma that is sometimes confused with PN) were also found to have homozygous C16orf57 mutations. Given the role of the previous DC genes in telomere maintenance, telomere length was analysed in these patients and found to be comparable to age-matched controls. These findings suggest that mutations in C16orf57 unify a distinct set of families which clinically can be categorized as DC, PN or RTS. This study also highlights the multi-system nature (wider than just poikiloderma and neutropenia) of the clinical features of affected individuals (and therefore house-keeping function of C16orf57), a possible role for C16orf57 in apoptosis, as well as a distinct difference from previously characterized DC patients because telomere length was normal

Germline ERCC excision repair 6 like 2 (ERCC6L2) mutations lead to impaired erythropoiesis and reshaping of the bone marrow microenvironment

Despite the inclusion of inherited myeloid malignancies as a separate entity in the World Health Organization Classification, many established predisposing loci continue to lack functional characterization. While germline mutations in the DNA repair factor ERCC excision repair 6 like 2 (ERCC6L2) give rise to bone marrow failure and acute myeloid leukaemia, their consequences on normal haematopoiesis remain unclear. To functionally characterise the dual impact of germline ERCC6L2 loss on human primary haematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs), we challenged ERCC6L2-silenced and patient-derived cells ex vivo. Here, we show for the first time that ERCC6L2-deficiency in HSPCs significantly impedes their clonogenic potential and leads to delayed erythroid differentiation. This observation was confirmed by CIBERSORTx RNA-sequencing deconvolution performed on ERCC6L2-silenced erythroid-committed cells, which demonstrated higher proportions of polychromatic erythroblasts and reduced orthochromatic erythroblasts versus controls. In parallel, we demonstrate that the consequences of ERCC6L2-deficiency are not limited to HSPCs, as we observe a striking phenotype in patient-derived and ERCC6L2-silenced MSCs, which exhibit enhanced osteogenesis and suppressed adipogenesis. Altogether, our study introduces a valuable surrogate model to study the impact of inherited myeloid mutations and highlights the importance of accounting for the influence of germline mutations in HSPCs and their microenvironment.Peer reviewe

DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation

This work was funded by The Medical Research Council UK (MR/K000292/1), Children with Cancer UK (2013/144), Barts and The London Charity (845/1796), and Leukaemia Lymphoma Research/Bloodwise (14032)

Differences in Disease Severity but Similar Telomere Lengths in Genetic Subgroups of Patients with Telomerase and Shelterin Mutations

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Homozygous OB-fold variants in telomere protein TPP1 are associated with dyskeratosis congenita–like phenotypes

This research was originally published in Blood Online. Tummala, H., et al. (2018). "Homozygous OB-fold variants in telomere protein TPP1 are associated with dyskeratosis congenita–like phenotypes." Blood 132(12): 1349-1353. Title. Blood. Prepublished Sept 20 2018; DOI https://doi.org/10.1182/blood-2018-03-837799Dyskeratosis congenita (DC) and its severe form, Hoyeraal-Hreidarsson syndrome (HHS), are rare and have life-threatening failure of hematopoiesis. Typically, DC patients present with disease features such as nail dystrophy, oral leukoplakia, and abnormal skin pigmentation along with peripheral pancytopenia and marrow hypoplasia with strong predisposition to cancer.1 In DC, hematopoietic failure occurs due to critical shortening of telomeres,2,3 which enhances the DNA damage response4,5 and leads to premature senescence of hematopoietic stem cellsMedical Research Council (grant MR/P018440/1), Bloodwise (grant 14032), and Children with Cancer UK (grant 2013/144)

Studies on the basis of the inherited bone marrow failure syndromes: Fanconi's anaemia and dyskeratosis congenita.

A number of inherited disorders are associated with bone marrow failure. Amongst them Fanconi's anaemia (FA) is the most common and, together with dyskeratosis congenita (DC), the best characterized. The aim of this study was twofold: A. To devise a strategy which may prermit "expression cloning" of the FA genes. B: To characterise cytogenetic (and molecular) features of cells from FA and DC patients which may provide a better understanding of the basis of these disorders. As a first step towards cloning the gene (s) for Fanconi's anaemia (FA) I devised a selection system [based on the DNA cross-linking agents mitomycin-c (MMC) and diepoxybutane (DEB)] that discriminates between FA and normal cells. 'Mixing experiments' (where approximately 10 normal cells were co-plated with 106 FA cells) demonstrated that it is possible to kill FA cells at high density without significantly affecting the cloning efficiency of normal cells. Transfection of FA fibroblasts with normal DNA (mouse genomic, human genomic, and human cDNA) either by calcium phosphate precipitation or by electroporation yielded 11 DEB and MMC resistant colonies. However, southern analysis of the DNA from these colonies with the appropriate probes gave no positive signal, and thus no "handle" to recover the FA gene (s). Experiments addressing the effect of the specific DNA topoisomerase I inhibitor, camptothecin, on FA and normal cells showed: 1. The FA lymphocytes have increased chromosomal breakage compared to normal lymphocytes after incubation with camptothecin (p=0.006). 2. Incubation of peripheral blood lymphocytes (pbl) from normal subjects with camptothecin, produced the same type of chromosomal breakage as that seen in FA lymphocytes. 3. FA fibroblasts were more sensitive to camptothecin than normal fibroblasts. These data are compatible with either a defect which makes topoisomerase I more crucial, or its function being abnormal in some FA patients. In patients with DC, primary skin fibroblast cultures were abnormal in both morphology and growth rate. Survival studies using 4 clastogens and gamma-irradiation showed no significant difference between DC and normal fibroblasts. Cytogenetic studies performed on pbl showed no difference between DC and normal lymphocytes with or without prior incubation with clastogens. However, bone marrow from 1 out of 3 patients and fibroblasts from 2 out of 4 patients showed numerous unbalanced chromosomal rearrangements in the absence of clastogenic agents. Although patients with FA and DC share some features in common they appear to differ in two fundamental ways: Firstly, unlike Fanconi cells, DC cells are not hypersensitive to clastogens. Secondly the primary defect in DC appears to predipose cells to developing chromosomal rearrangements rather than to chromosomal gaps and breaks seen in FA