RESUMO
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.
Assuntos
Transtornos da Insuficiência da Medula Óssea/diagnóstico , Disceratose Congênita/diagnóstico , Retardo do Crescimento Fetal/diagnóstico , Triagem de Portadores Genéticos/métodos , Deficiência Intelectual/diagnóstico , Microcefalia/diagnóstico , Telômero/patologia , Adolescente , Adulto , Fatores Etários , Idoso , Doenças Assintomáticas , Transtornos da Insuficiência da Medula Óssea/genética , Transtornos da Insuficiência da Medula Óssea/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Disceratose Congênita/genética , Disceratose Congênita/patologia , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Heterozigoto , Humanos , Lactente , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Microcefalia/genética , Microcefalia/patologia , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Análise de Sobrevida , Telômero/metabolismo , Homeostase do TelômeroRESUMO
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.
Assuntos
Transtornos da Insuficiência da Medula Óssea/genética , Mutação da Fase de Leitura/genética , Fator de Transcrição Sp1/genética , Transcrição Gênica/genética , Feminino , Humanos , Masculino , Linhagem , Transcriptoma/genética , Regulação para Cima/genética , Dedos de Zinco/genéticaRESUMO
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.
Assuntos
Mutação em Linhagem Germinativa , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Desaminase/genética , Dineínas do Axonema/genética , Estudos de Coortes , Humanos , Degradação do RNAm Mediada por Códon sem Sentido , Linhagem , Perforina/genética , Glicoproteínas da Membrana de Plaquetas/genética , RNA Helicases/genética , Receptores de Interleucina-17/genética , Proteínas de Transporte Vesicular/genética , Sequenciamento do ExomaAssuntos
Aminopeptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Disceratose Congênita/genética , Mutação Puntual , Serina Proteases/genética , Complexo Shelterina , Proteínas de Ligação a Telômeros , Adulto , Aminopeptidases/química , Criança , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Feminino , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Conformação Proteica , Serina Proteases/química , Complexo Shelterina/genética , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/genéticaRESUMO
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.
Assuntos
Alelos , Doenças da Medula Óssea/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Doenças Genéticas Inatas/metabolismo , Instabilidade Genômica , Transcrição Gênica , Células A549 , Doenças da Medula Óssea/genética , Doenças da Medula Óssea/patologia , DNA Helicases/genética , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Células HeLa , Humanos , Masculino , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , SíndromeAssuntos
Estudos de Associação Genética , Variação Genética , Doenças Hematológicas/complicações , Doenças Hematológicas/diagnóstico , Fenótipo , Rádio (Anatomia)/anormalidades , Sinostose/diagnóstico , Sinostose/genética , Ulna/anormalidades , Feminino , Proteínas de Homeodomínio/genética , Humanos , Proteína do Locus do Complexo MDS1 e EVI1/genética , Masculino , Mutação , Linhagem , Rádio (Anatomia)/diagnóstico por imagem , Sinostose/complicações , Ulna/diagnóstico por imagemRESUMO
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.
Assuntos
Disceratose Congênita/diagnóstico , Disceratose Congênita/genética , Exoma/genética , Variação Genética/genética , DNA Ligase Dependente de ATP/genética , Proteínas de Ligação a DNA/genética , Humanos , Linhagem , Diester Fosfórico Hidrolases/genética , Análise de Sequência de DNA , Síndrome , Fatores de Transcrição/genéticaRESUMO
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.
