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.

The clinical picture of ERCC6L2 disease: from bone marrow failure to acute leukemia.

Biallelic germ line excision repair cross-complementing 6 like 2 (ERCC6L2) variants strongly predispose to bone marrow failure (BMF) and myeloid malignancies, characterized by somatic TP53-mutated clones and erythroid predominance. We present a series of 52 subjects (35 families) with ERCC6L2 biallelic germ line variants collected retrospectively from 11 centers globally, with a follow-up of 1165 person-years. At initial investigations, 32 individuals were diagnosed with BMF and 15 with a hematological malignancy (HM). The subjects presented with 19 different variants of ERCC6L2, and we identified a founder mutation, c.1424delT, in Finnish patients. The median age of the subjects at baseline was 18 years (range, 2-65 years). Changes in the complete blood count were mild despite severe bone marrow (BM) hypoplasia and somatic TP53 mutations, with no significant difference between subjects with or without HMs. Signs of progressive disease included increasing TP53 variant allele frequency, dysplasia in megakaryocytes and/or erythroid lineage, and erythroid predominance in the BM morphology. The median age at the onset of HM was 37.0 years (95% CI, 31.5-42.5; range, 12-65 years). The overall survival (OS) at 3 years was 95% (95% CI, 85-100) and 19% (95% CI, 0-39) for patients with BMF and HM, respectively. Patients with myelodysplastic syndrome or acute myeloid leukemia with mutated TP53 undergoing hematopoietic stem cell transplantation had a poor outcome with a 3-year OS of 28% (95% CI, 0-61). Our results demonstrated the importance of early recognition and active surveillance in patients with biallelic germ line ERCC6L2 variants.

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.

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.

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.

High-throughput STELA provides a rapid test for the diagnosis of telomere biology disorders.

Telomere biology disorders are complex clinical conditions that arise due to mutations in genes required for telomere maintenance. Telomere length has been utilised as part of the diagnostic work-up of patients with these diseases; here, we have tested the utility of high-throughput STELA (HT-STELA) for this purpose. HT-STELA was applied to a cohort of unaffected individuals (n = 171) and a retrospective cohort of mutation carriers (n = 172). HT-STELA displayed a low measurement error with inter- and intra-assay coefficient of variance of 2.3% and 1.8%, respectively. Whilst telomere length in unaffected individuals declined as a function of age, telomere length in mutation carriers appeared to increase due to a preponderance of shorter telomeres detected in younger individuals (< 20 years of age). These individuals were more severely affected, and age-adjusted telomere length differentials could be used to stratify the cohort for overall survival (Hazard Ratio = 5.6 (1.5-20.5); p < 0.0001). Telomere lengths of asymptomatic mutation carriers were shorter than controls (p < 0.0001), but longer than symptomatic mutation carriers (p < 0.0001) and telomere length heterogeneity was dependent on the diagnosis and mutational status. Our data show that the ability of HT-STELA to detect short telomere lengths, that are not readily detected with other methods, means it can provide powerful diagnostic discrimination and prognostic information. The rapid format, with a low measurement error, demonstrates that HT-STELA is a new high-quality laboratory test for the clinical diagnosis of an underlying telomeropathy.

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.

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

A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit.

The Human Phenotype Ontology in 2017.

Deep phenotyping has been defined as the precise and comprehensive analysis of phenotypic abnormalities in which the individual components of the phenotype are observed and described. The three components of the Human Phenotype Ontology (HPO; www.human-phenotype-ontology.org) project are the phenotype vocabulary, disease-phenotype annotations and the algorithms that operate on these. These components are being used for computational deep phenotyping and precision medicine as well as integration of clinical data into translational research. The HPO is being increasingly adopted as a standard for phenotypic abnormalities by diverse groups such as international rare disease organizations, registries, clinical labs, biomedical resources, and clinical software tools and will thereby contribute toward nascent efforts at global data exchange for identifying disease etiologies. This update article reviews the progress of the HPO project since the debut Nucleic Acids Research database article in 2014, including specific areas of expansion such as common (complex) disease, new algorithms for phenotype driven genomic discovery and diagnostics, integration of cross-species mapping efforts with the Mammalian Phenotype Ontology, an improved quality control pipeline, and the addition of patient-friendly terminology.

Phenopolis: an open platform for harmonization and analysis of genetic and phenotypic data.

SUMMARY: Phenopolis is an open-source web server providing an intuitive interface to genetic and phenotypic databases. It integrates analysis tools such as variant filtering and gene prioritization based on phenotype. The Phenopolis platform will accelerate clinical diagnosis, gene discovery and encourage wider adoption of the Human Phenotype Ontology in the study of rare genetic diseases. AVAILABILITY AND IMPLEMENTATION: A demo of the website is available at https://phenopolis.github.io . If you wish to install a local copy, source code and installation instruction are available at https://github.com/phenopolis . The software is implemented using Python, MongoDB, HTML/Javascript and various bash shell scripts. CONTACT: n.pontikos@ucl.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ERCC6L2 mutations link a distinct bone-marrow-failure syndrome to DNA repair and mitochondrial function.

Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knockdown cells and abolished ERCCGL2 traffic to the mitochondria and nucleus in response to this DNA-damaging agent. Collectively, these observations identify a distinct bone-marrow-failure syndrome due to mutations in ERCC6L2, a gene implicated in DNA repair and mitochondrial function.

Constitutional mutations in RTEL1 cause severe dyskeratosis congenita.

Dyskeratosis congenita (DC) and its phenotypically severe variant, Hoyeraal-Hreidarsson syndrome (HHS), are multisystem bone-marrow-failure syndromes in which the principal pathology is defective telomere maintenance. The genetic basis of many cases of DC and HHS remains unknown. Using whole-exome sequencing, we identified biallelic mutations in RTEL1, encoding a helicase essential for telomere maintenance and regulation of homologous recombination, in an individual with familial HHS. Additional screening of RTEL1 identified biallelic mutations in 6/23 index cases with HHS but none in 102 DC or DC-like cases. All 11 mutations in ten HHS individuals from seven families segregated in an autosomal-recessive manner, and telomere lengths were significantly shorter in cases than in controls (p = 0.0003). This group had significantly higher levels of telomeric circles, produced as a consequence of incorrect processing of telomere ends, than did controls (p = 0.0148). These biallelic RTEL1 mutations are responsible for a major subgroup (∼29%) of HHS. Our studies show that cells harboring these mutations have significant defects in telomere maintenance, but not in homologous recombination, and that incorrect resolution of T-loops is a mechanism for telomere shortening and disease causation in humans. They also demonstrate the severe multisystem consequences of its dysfunction.

Exome sequencing identifies autosomal-dominant SRP72 mutations associated with familial aplasia and myelodysplasia.

Aplastic anemia (AA) and myelodysplasia (MDS) are forms of bone marrow failure that are often part of the same progressive underlying disorder. While most cases are simplex and idiopathic, some show a clear pattern of inheritance; therefore, elucidating the underlying genetic cause could lead to a greater understanding of this spectrum of disorders. We used a combination of exome sequencing and SNP haplotype analysis to identify causative mutations in a family with a history of autosomal-dominant AA/MDS. We identified a heterozygous mutation in SRP72, a component of the signal recognition particle (SRP) that is responsible for the translocation of nascent membrane-bound and excreted proteins to the endoplasmic reticulum. A subsequent screen revealed another autosomal-dominant family with an inherited heterozygous SRP72 mutation. Transfection of these sequences into mammalian cells suggested that these proteins localize incorrectly within the cell. Furthermore, coimmunoprecipitation of epitope-tagged SRP72 indicated that the essential RNA component of the SRP did not fully associate with one of the SRP72 variants. These results suggest that inherited mutations in a component of the SRP have a role in the pathophysiology of AA/MDS, identifying a third pathway for developing these disorders alongside transcription factor and telomerase mutations.

Revertant somatic mosaicism by mitotic recombination in dyskeratosis congenita.

Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.

Inflammatory skin and bowel disease linked to ADAM17 deletion.

We performed genetic and immunohistochemical studies in a sister and brother with autosomal recessive neonatal inflammatory skin and bowel lesions. The girl died suddenly at 12 years of age from parvovirus B19-associated myocarditis; her brother had mild cardiomyopathy. We identified a loss-of-function mutation in ADAM17, which encodes a disintegrin and metalloproteinase 17 (also called tumor necrosis factor α [TNF-α]-converting enzyme, or TACE), as the probable cause of this syndrome. Peripheral-blood mononuclear cells (PBMCs) obtained from the brother at 17 years of age showed high levels of lipopolysaccharide-induced production of interleukin-1ß and interleukin-6 but impaired release of TNF-α. Despite repeated skin infections, this young man has led a relatively normal life. (Funded by Barts and the London Charity and the European Commission Seventh Framework Programme.).

Mutations in the telomere capping complex in bone marrow failure and related syndromes.

Dyskeratosis congenita and its variants have overlapping phenotypes with many disorders including Coats plus, and their underlying pathology is thought to be one of defective telomere maintenance. Recently, biallelic CTC1 mutations have been described in patients with syndromes overlapping Coats plus. CTC1, STN1 and TEN1 are part of the telomere-capping complex involved in maintaining telomeric structural integrity. Based on phenotypic overlap we screened 73 genetically uncharacterized patients with dyskeratosis congenita and related bone marrow failure syndromes for mutations in this complex. Biallelic CTC1 mutations were identified in 6 patients but none in either STN1 or TEN1. We have expanded the phenotypic spectrum associated with CTC1 mutations and report that intracranial and retinal abnormalities are not a defining feature, as well as showing that the effect of these mutations on telomere length is variable. The study also demonstrates the lack of disease-causing mutations in other components of the telomere-capping complex.