The evolving genetic landscape of telomere biology disorder dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as 'DC-like' (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients' blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders.

Ribosomal DNA copy number is associated with body mass in humans and other mammals.

Body mass results from a complex interplay between genetics and environment. Previous studies of the genetic contribution to body mass have excluded repetitive regions due to the technical limitations of platforms used for population scale studies. Here we apply genome-wide approaches, identifying an association between adult body mass and the copy number (CN) of 47S-ribosomal DNA (rDNA). rDNA codes for the 18 S, 5.8 S and 28 S ribosomal RNA (rRNA) components of the ribosome. In mammals, there are hundreds of copies of these genes. Inter-individual variation in the rDNA CN has not previously been associated with a mammalian phenotype. Here, we show that rDNA CN variation associates with post-pubertal growth rate in rats and body mass index in adult humans. rDNA CN is not associated with rRNA transcription rates in adult tissues, suggesting the mechanistic link occurs earlier in development. This aligns with the observation that the association emerges by early adulthood.

PSIP1/LEDGF reduces R-loops at transcription sites to maintain genome integrity.

R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.

Transcriptome Analysis Reveals Vimentin-Induced Disruption of Cell-Cell Associations Augments Breast Cancer Cell Migration.

In advanced metastatic cancers with reduced patient survival and poor prognosis, expression of vimentin, a type III intermediate filament protein is frequently observed. Vimentin appears to suppress epithelial characteristics and augments cell migration but the molecular basis for these changes is not well understood. Here, we have ectopically expressed vimentin in MCF-7 and investigated its genomic and functional implications. Vimentin changed the cell shape by decreasing major axis, major axis angle and increased cell migration, without affecting proliferation. Vimentin downregulated major keratin genes KRT8, KRT18 and KRT19. Transcriptome-coupled GO and KEGG analyses revealed that vimentin-affected genes were linked to either cell-cell/cell-ECM or cell cycle/proliferation specific pathways. Using shRNA mediated knockdown of vimentin in two cell types; MCF-7FV (ectopically expressing) and MDA-MB-231 (endogenously expressing), we identified a vimentin-specific signature consisting of 13 protein encoding genes (CDH5, AXL, PTPRM, TGFBI, CDH10, NES, E2F1, FOXM1, CDC45, FSD1, BCL2, KIF26A and WISP2) and two long non-coding RNAs, LINC00052 and C15ORF9-AS1. CDH5, an endothelial cadherin, which mediates cell-cell junctions, was the most downregulated protein encoding gene. Interestingly, downregulation of CDH5 by shRNA significantly increased cell migration confirming our RNA-Seq data. Furthermore, presence of vimentin altered the lamin expression in MCF-7. Collectively, we demonstrate, for the first time, that vimentin in breast cancer cells could change nuclear architecture by affecting lamin expression, which downregulates genes maintaining cell-cell junctions resulting in increased cell migration.

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.

The biology and management of dyskeratosis congenita and related disorders of telomeres.

BACKGROUND: Dyskeratosis congenita (DC) is a multisystem syndrome characterized by mucocutaneous abnormalities, bone marrow failure, and predisposition to cancer. Studies over the last 25 years have led to the identification of 18 disease genes. These have a principal role in telomere maintenance, and patients usually have very short/abnormal telomeres. The advances have also led to the unification of DC with a number of other diseases, now collectively referred to as the telomeropathies or telomere biology disorders. WHAT IS COVERED: Clinical features, genetics, and biology of the different subtypes. Expert view on diagnosis, treatment of the hematological complications and future. EXPERT VIEW: As these are very pleotropic disorders affecting multiple organs, a high index of suspicion is necessary to make the diagnosis. Telomere length measurement and genetic analysis of the disease genes have become useful diagnostic tools. Although hematological defects can respond to danazol/oxymetholone, the only current curative treatment for these is hematopoietic stem cell transplantation (HSCT) using fludarabine-based conditioning protocols. New therapies are needed where danazol/oxymetholone is ineffective and HSCT is not feasible.

Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone-marrow-failure disorder characterized by a triad of mucocutaneous features that include abnormal skin pigmentation, nail dystrophy, and oral leucoplakia. Despite the identification of several genetic variants that cause DC, a significant proportion of probands remain without a molecular diagnosis. In a cohort of eight independent DC-affected families, we have identified a remarkable series of heterozygous germline variants in the gene encoding thymidylate synthase (TYMS). Although the inheritance appeared to be autosomal recessive, one parent in each family had a wild-type TYMS coding sequence. Targeted genomic sequencing identified a specific haplotype and rare variants in the naturally occurring TYMS antisense regulator ENOSF1 (enolase super family 1) inherited from the other parent. Lymphoblastoid cells from affected probands have severe TYMS deficiency, altered cellular deoxyribonucleotide triphosphate pools, and hypersensitivity to the TYMS-specific inhibitor 5-fluorouracil. These defects in the nucleotide metabolism pathway resulted in genotoxic stress, defective transcription, and abnormal telomere maintenance. Gene-rescue studies in cells from affected probands revealed that post-transcriptional epistatic silencing of TYMS is occurring via elevated ENOSF1. These cell and molecular abnormalities generated by the combination of germline digenic variants at the TYMS-ENOSF1 locus represent a unique pathogenetic pathway for DC causation in these affected individuals, whereas the parents who are carriers of either of these variants in a singular fashion remain unaffected.

Inherited bone marrow failure in the pediatric patient.

Inherited bone marrow (BM) failure syndromes are a diverse group of disorders characterized by BM failure, usually in association with ≥1 extrahematopoietic abnormalities. BM failure, which can involve ≥1 cell lineages, often presents in the pediatric age group. Furthermore, some children initially labeled as having idiopathic aplastic anemia or myelodysplasia represent cryptic cases of inherited BM failure. Significant advances in the genetics of these syndromes have been made, identifying more than 100 disease genes, giving insights into normal hematopoiesis and how it is disrupted in patients with BM failure. They have also provided important information on fundamental biological pathways, including DNA repair: Fanconi anemia (FA) genes; telomere maintenance: dyskeratosis congenita (DC) genes; and ribosome biogenesis: Shwachman-Diamond syndrome and Diamond-Blackfan anemia genes. In addition, because these disorders are usually associated with extrahematopoietic abnormalities and increased risk of cancer, they have provided insights into human development and cancer. In the clinic, genetic tests stemming from the recent advances facilitate diagnosis, especially when clinical features are insufficient to accurately classify a disorder. Hematopoietic stem cell transplantation using fludarabine-based protocols has significantly improved outcomes, particularly in patients with FA or DC. Management of some other complications, such as cancer, remains a challenge. Recent studies have suggested the possibility of new and potentially more efficacious therapies, including a renewed focus on hematopoietic gene therapy and drugs [transforming growth factor-ß inhibitors for FA and PAPD5, a human poly(A) polymerase, inhibitors for DC] that target disease-specific defects.

Genome-wide whole-blood transcriptome profiling across inherited bone marrow failure subtypes.

Gene expression profiling has long been used in understanding the contribution of genes and related pathways in disease pathogenesis and susceptibility. We have performed whole-blood transcriptomic profiling in a subset of patients with inherited bone marrow failure (IBMF) whose diseases are clinically and genetically characterized as Fanconi anemia (FA), Shwachman-Diamond syndrome (SDS), and dyskeratosis congenita (DC). We hypothesized that annotating whole-blood transcripts genome wide will aid in understanding the complexity of gene regulation across these IBMF subtypes. Initial analysis of these blood-derived transcriptomes revealed significant skewing toward upregulated genes in patients with FA when compared with controls. Patients with SDS or DC also showed similar skewing profiles in their transcriptional status revealing a common pattern across these different IBMF subtypes. Gene set enrichment analysis revealed shared pathways involved in protein translation and elongation (ribosome constituents), RNA metabolism (nonsense-mediated decay), and mitochondrial function (electron transport chain). We further identified a discovery set of 26 upregulated genes at stringent cutoff (false discovery rate < 0.05) that appeared as a unified signature across the IBMF subtypes. Subsequent transcriptomic analysis on genetically uncharacterized patients with BMF revealed a striking overlap of genes, including 22 from the discovery set, which indicates a unified transcriptional drive across the classic (FA, SDS, and DC) and uncharacterized BMF subtypes. This study has relevance in disease pathogenesis, for example, in explaining the features (including the BMF) common to all patients with IBMF and suggests harnessing this transcriptional signature for patient benefit.

A frameshift variant in specificity protein 1 triggers superactivation of Sp1-mediated transcription in familial bone marrow failure.

Inherited bone marrow failure (BMF) syndromes are a heterogeneous group of diseases characterized by defective hematopoiesis and often predisposing to myelodysplastic syndrome (MDS) and acute myelogenous leukemia. We have studied a large family consisting of several affected individuals with hematologic abnormalities, including one family member who died of acute leukemia. By whole-exome sequencing, we identified a novel frameshift variant in the ubiquitously expressed transcription factor specificity protein 1 (SP1). This heterozygous variant (c.1995delA) truncates the canonical Sp1 molecule in the highly conserved C-terminal DNA-binding zinc finger domains. Transcriptomic analysis and gene promoter characterization in patients' blood revealed a hypermorphic effect of this Sp1 variant, triggering superactivation of Sp1-mediated transcription and driving significant up-regulation of Sp1 target genes. This familial genetic study indicates a central role for Sp1 in causing autosomal dominant transmission of BMF, thereby confirming its critical role in hematopoiesis in humans.

The complex genetic landscape of familial MDS and AML reveals pathogenic germline variants.

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.

Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants.

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients' lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients' LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients' LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA-RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

Marked overlap of four genetic syndromes with dyskeratosis congenita confounds clinical diagnosis.

Dyskeratosis congenita is a highly pleotropic genetic disorder. This heterogeneity can lead to difficulties in making an accurate diagnosis and delays in appropriate management. The aim of this study was to determine the underlying genetic basis in patients presenting with features of dyskeratosis congenita and who were negative for mutations in the classical dyskeratosis congenita genes. By whole exome and targeted sequencing, we identified biallelic variants in genes that are not associated with dyskeratosis congenita in 17 individuals from 12 families. Specifically, these were homozygous variants in USB1 (8 families), homozygous missense variants in GRHL2 (2 families) and identical compound heterozygous variants in LIG4 (2 families). All patients had multiple somatic features of dyskeratosis congenita but not the characteristic short telomeres. Our case series shows that biallelic variants in USB1, LIG4 and GRHL2, the genes mutated in poikiloderma with neutropenia, LIG4/Dubowitz syndrome and the recently recognized ectodermal dysplasia/short stature syndrome, respectively, cause features that overlap with dyskeratosis congenita. Strikingly, these genes also overlap in their biological function with the known dyskeratosis congenita genes that are implicated in telomere maintenance and DNA repair pathways. Collectively, these observations demonstrate the marked overlap of dyskeratosis congenita with four other genetic syndromes, confounding accurate diagnosis and subsequent management. This has important implications for establishing a genetic diagnosis when a new patient presents in the clinic. Patients with clinical features of dyskeratosis congenita need to have genetic analysis of USB1, LIG4 and GRHL2 in addition to the classical dyskeratosis congenita genes and telomere length measurements.