Disruption of telomerase trafficking by TCAB1 mutation causes dyskeratosis congenita

Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought to precipitate DC by reducing either the catalytic activity or the overall levels of the telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a telomerase holoenzyme protein that facilitates trafficking of telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt telomerase localization to Cajal bodies, resulting in misdirection of telomerase RNA to nucleoli, which prevents telomerase from elongating telomeres. Our findings establish telomerase mislocalization as a novel cause of DC, and suggest that telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.     

Fluorescence microscopy methods for examining telomeres during cell aging

Telomeres are protective structures, composed of nucleic acids and a complex protein mixture, located at the end of the chromosomes. They play an important role in preventing genomic instability and ensuring cell health. Defects in telomere integrity result in cell dysfunction and the development of diseases, including neurodegenerative disorders, cancer and premature aging syndromes, among others. Loss of telomere integrity during normal cell aging also initiates DNA damage signals that culminate in the senescence phenotype. Fluorescence microscopy has allowed researchers to study the dynamics, shape, localization, and co-distribution of telomeres with proteins of interest. The microscopy tools to investigate these structures have evolved, making it possible to understand in greater detail the molecular mechanisms affecting telomeres that contribute to cell aging and the development of age-related diseases. Using human fibroblasts as an example, we will highlight several characteristics of telomeres that can be investigated using three different microscopy systems, including wide-field microscopy, and the two super-resolution techniques called 3D Structured Illumination Microscopy (3D-SIM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). In this review, we will also discuss their limitations and highlight their importance in answering telomere-related scientific questions.     

Engineered telomere degradation models dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4-5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can be modeled in the mouse.     

Replicative senescence and senescence-like state induced in cancer-derived cells

Studies on the replicative senescence and premature senescence induced by various stresses in normal somatic cells have provided important clues on the role of telomere shortening and mechanisms involved in aging processes and carcinogenesis. Recent work revealed that cancer cells also are induced to undergo replicative senescence state via telomere shortening as well as to enter a senescence-like state by the activation of cell cycle inhibitory pathways. Although less relevant in terms of aging physiology, studies on these phenomena in cancer cells have yielded important information on telomerase regulation and the roles of tumor suppressors in senescence and immortalization, and are expected to generate valuable anti-cancer strategies. Several features of the phenotypes specific for the senescent and senescence-like states induced in cancer cells are discussed.     

Role of telomere dysfunction in aging and its detection by biomarkers

Aging is a complex process that has been shown to be linked to accumulation of DNA damage. Telomere shortening represents a cell-intrinsic mechanism leading to DNA damage accumulation and activation of DNA damage checkpoints in aging cells. Activation of DNA damage checkpoints in response to telomere dysfunction results in induction of cellular senescence—a permanent cell cycle arrest. Senescence represents a tumor suppressor mechanism protecting cells from evolution of genomic instability and transformation. As a drawback, telomere shortening may also limit tissue renewal and regenerative capacity of tissues in response to aging and chronic disease. In aged organs, telomere shortening may also increase the cancer risk by initiation of chromosomal instability, loss of proliferative competition of aging stem cells, and selection of aberrant growing clones. Consequently, aged individuals are more susceptible and vulnerable to various diseases and show an increased cancer risk. Recently, proteins were discovered, which are induced by telomere dysfunction and DNA damage. It was shown that these proteins represent new biomarkers of human aging and disease. Here, we review the scientific background and experimental data on these newly discovered biomarkers.     

Growth,telomere dynamics and successful and unsuccessful human aging

This paper links mass trajectories with telomere dynamics to construct theoretical models of successful and unsuccessful aging in human beings. It couples parameters of telomere length in somatic cells, as expressed by the terminal restriction fragment (TRF), at birth and the rate of telomere attrition thereafter with nonlinear models of somatic growth to predict the probability of surviving disease free, based on the assumption that telomere length in replicating somatic cells is a surrogate indicator of aging determinants in humans. The models capture aspects of individual variation in successful and unsuccessful aging and the long-term consequences of rapid growth early in life.     

Elevated telomere-telomere recombination in WRN-deficient, telomere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway

Werner Syndrome (WS) is characterized by premature aging, genomic instability, and cancer. The combined impact of WRN helicase deficiency and limiting telomere reserves is central to disease pathogenesis. Here, we report that cells doubly deficient for telomerase and WRN helicase show chromosomal aberrations and elevated recombination rates between telomeres of sister chromatids. Somatic reconstitution of WRN function, but not a WRN helicase-deficient mutant, abolished telomere sister chromatid exchange (T-SCE), indicating that WRN normally represses T-SCEs. Elevated T-SCE was associated with greater immortalization potential and resultant tumors maintained telomeres via the alternative lengthening of telomere (ALT) pathway. We propose that the increased incidence of chromosomal instability and cancer in WS relates in part to aberrant recombinations between sister chromatids at telomeres, which facilitates the activation of ALT and engenders cancer-relevant chromosomal aberrations and tumor formation.     

Telomere Phenotypes in Females with Heterozygous Mutations in the Dyskeratosis Congenita 1 (DKC1) Gene

Dyskeratosis congenita (DC) is a telomere‐mediated syndrome defined by mucocutaneous features. The X‐linked mode of inheritance accounts for half the cases, and is thought to predominantly manifest in childhood as bone marrow failure. We identified two male probands who presented in the fifth decade with idiopathic pulmonary fibrosis and cancer. Their pedigrees displayed consecutively affected generations. Five of six females (83%) manifested mucocutaneous features of DC, and two had wound‐healing complications. No mutations in autosomal dominant telomere genes were present, but exome sequencing revealed novel variants in the X‐chromosome DKC1 gene that predicted missense mutations in conserved residues, p.Thr49Ser and p.Pro409Arg. Variants segregated with the telomere phenotype, and affected females were heterozygotes, showing skewed X‐inactivation. Telomerase RNA levels were compromised in cells from DKC1 mutation carriers, consistent with their pathogenic role. These findings indicate that females with heterozygous DKC1 mutations may be at increased risk for developing penetrant telomere phenotypes that, at times, may be associated with clinical morbidity.     

The role of telomere biology in bone marrow failure and other disorders

Telomeres, consisting of nucleotide repeats and a protein complex at chromosome ends, are essential in maintaining chromosomal integrity. Dyskeratosis congenita (DC) is the inherited bone marrow failure syndrome (IBMFS) that epitomizes the effects of abnormal telomere biology. Patients with DC have extremely short telomere lengths (<1st percentile) and many have mutations in telomere biology genes. Interpretation of telomere length in other IBMFSs is less straightforward. Abnormal telomere shortening has been reported in patients with apparently acquired hematologic disorders, including aplastic anemia, myeolodysplasia, paroxysmal nocturnal hemoglobinuria, and leukemia. In these disorders, the shortest-lived cells have the shortest telomeres, suggestive of increased hematopoietic stress. Telomeres are also markers of replicative and/or oxidative stress in other complex disease pathways, such as inflammation, stress, and carcinogenesis. The spectrum of related disorders caused by mutations in telomere biology genes extends beyond classical DC to include marrow failure that does not respond to immunosuppression, idiopathic pulmonary fibrosis, and possibly other syndromes. We suggest that such patients be categorized as having an inherited disorder of telomere biology. Longitudinal studies of patients with very short telomeres but without classical DC are necessary to further understand the long-term sequelae, such as malignancy, osteonecrosis/osteoporosis, and pulmonary and liver disease.     

Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita

Dyskeratosis congenita (DC) patients suffer a progressive and ultimately fatal loss of hematopoietic renewal correlating with critically short telomeres. The predominant X-linked form of DC results from substitutions in dyskerin, a protein required both for ribosomal RNA (rRNA) pseudouridine modification and for cellular accumulation of telomerase RNA (TER). Accordingly, alternative models have posited that the exhaustion of cellular renewal in X-linked DC arises as a primary consequence of ribosome deficiency or telomerase deficiency. Here we test, for the first time, whether X-linked DC patient cells are compromised for telomerase function at telomeres. We show that telomerase activation in family-matched control cells allows telomere elongation and telomere length maintenance, while telomerase activation in X-linked DC patient cells fails to prevent telomere erosion with proliferation. Furthermore, we demonstrate by phenotypic rescue that telomere defects in X-linked DC patient cells arise solely from reduced accumulation of TER. We also show that X-linked DC patient cells averted from premature senescence support normal levels of rRNA pseudouridine modification and normal kinetics of rRNA precursor processing, in contrast with phenotypes reported for a proposed mouse model of the human disease. These findings support the significance of telomerase deficiency in the pathology of X-linked DC.     

Complex phenotype of dyskeratosis congenita and mood dysregulation with novel homozygous RTEL1 and TPH1 variants

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome caused by germline mutations in telomere biology genes. Patients have extremely short telomeres for their age and a complex phenotype including oral leukoplakia, abnormal skin pigmentation, and dysplastic nails in addition to bone marrow failure, pulmonary fibrosis, stenosis of the esophagus, lacrimal ducts and urethra, developmental anomalies, and high risk of cancer. We evaluated a patient with features of DC, mood dysregulation, diabetes, and lack of pubertal development. Family history was not available but genome‐wide genotyping was consistent with consanguinity. Whole exome sequencing identified 82 variants of interest in 80 genes based on the following criteria: homozygous, <0.1% minor allele frequency in public and in‐house databases, nonsynonymous, and predicted deleterious by multiple in silico prediction programs. Six genes were identified likely contributory to the clinical presentation. The cause of DC is likely due to homozygous splice site variants in regulator of telomere elongation helicase 1, a known DC and telomere biology gene. A homozygous, missense variant in tryptophan hydroxylase 1 may be clinically important as this gene encodes the rate limiting step in serotonin biosynthesis, a biologic pathway connected with mood disorders. Four additional genes (SCN4A, LRP4, GDAP1L1, and SPTBN5) had rare, missense homozygous variants that we speculate may contribute to portions of the clinical phenotype. This case illustrates the value of conducting detailed clinical and genomic evaluations on rare patients in order to identify new areas of research into the functional consequences of rare variants and their contribution to human disease.     

Enhanced telomere shortening in transformed lymphoblasts from patients with X linked dyskeratosis

AIM: Dyskeratosis congenita (DC) is characterised by the failure of those tissues that are rapidly dividing in the adult, particularly the skin, mucosae, and haemopoietic system. The X linked form of the disease is caused by mutations of the DKC1 gene, which encodes dyskerin, a protein that is necessary for the function of telomerase. Cultured DC lymphoblastoid cells are characterised by a reduced expansion of the cell population because of the progressive increase in apoptosis compared with the number of cell divisions. This report aimed to verify whether this is caused by a defect in telomerase function. METHODS: Variations in telomere length over time were evaluated in two cultured lymphoblastoid cell lines derived from patients with X linked DC and control cells derived from a non-affected individual. In addition, the effect of inhibiting poly (ADP-ribose) polymerase (PARP), which is involved in the cellular response to excessive telomere shortening, was assessed. One DC cell line and the control cells were treated with the specific PARP inhibitor 1,5-dihydroxyquinoline (IQ). RESULTS: In DC cells the increase in cell death was associated with progressive telomere shortening, and this was not seen in the control cells. Treatment with IQ delayed the increase of apoptosis in DC cells. CONCLUSIONS: These observations indicate that the reduced expansion that characterises cultured cells obtained from patients with X linked DC is caused by premature telomere shortening.     

Pharmacologic manipulation of the ataxia-telangiectasia mutated gene product as an intervention in age-related disease

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by progressive ataxia, elevated cancer incidence, and premature aging. A-T cells, Atm-deficient mice, and individuals with A-T show increased oxidant sensitivity, genomic instability, altered IGF-1 and p53 signaling, and rapid telomere shortening compared to normal controls. The gene mutated in A-T, ATM, regulates DNA repair, IGF-1 and p53 signaling, age pigment removal, antioxidant capacity, and telomere maintenance - pathways involved in and often attenuated with aging. Interestingly, flavonoids with chemopreventative effects, such as quercetin, genistein, and epigallocatechin gallate activate ATM. Since ATM activates pathways which increase genomic stability, oxidant resistance, and/or telomere stability, and since many diseases of old age (i.e., cancer, cardiovascular and neurodegenerative disease), result from attenuation of these pathways, pharmacologic manipulation of ATM activity via flavonoid intake may prove useful in slowing the appearance of age-associated disease.     

Telomere-associated aging disorders

Telomeres are dynamic nucleoprotein-DNA structures that cap and protect linear chromosome ends. Several monogenic inherited diseases that display features of human premature aging correlate with shortened telomeres, and are referred to collectively as telomeropathies. These disorders have overlapping symptoms and a common underlying mechanism of telomere dysfunction, but also exhibit variable symptoms and age of onset, suggesting they fall along a spectrum of disorders. Primary telomeropathies are caused by defects in the telomere maintenance machinery, whereas secondary telomeropathies have some overlapping symptoms with primary telomeropathies, but are generally caused by mutations in DNA repair proteins that contribute to telomere preservation. Here we review both the primary and secondary telomeropathies, discuss potential mechanisms for tissue specificity and age of onset, and highlight outstanding questions in the field and future directions toward elucidating disease etiology and developing therapeutic strategies.     

Dyskeratosis congenita: a genetic disorder of many faces

Dyskeratosis congenita (DC) is an inherited syndrome exhibiting marked clinical and genetic heterogeneity. It is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. Three genetic subtypes are recognized: X-linked recessive DC bears mutations in DKC1 , the gene encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein particles; autosomal dominant (AD) DC has heterozygous mutations in either TERC or TERT , the RNA and enzymatic components of telomerase, respectively, and autosomal recessive DC in which the genes involved remain largely elusive. Disease pathology is believed to be a consequence of chromosome instability because of telomerase deficiency due to mutations in DKC1 , TERC and TERT ; in patients with DKC1 mutations, defects in ribosomal RNA modification, ribosome biogenesis, translation control or mRNA splicing may also contribute to disease pathogenesis. The involvement of telomerase complex components in X-linked and AD forms and the presence of short telomeres in DC patients suggest that DC is primarily a disease of defective telomere maintenance. Treatment is variable and complicated by the development of secondary cancers but, being a monogenic disorder, it could potentially be treated by gene therapy. DC overlaps both clinically and genetically with several other diseases including Hoyeraal–Hreidarsson syndrome, aplastic anaemia and myelodysplasia, among others and its underlying telomeric defect has implications for a broader range of biological processes including ageing and many forms of cancer.     

Three novel truncating TINF2 mutations causing severe dyskeratosis congenita in early childhood

Dyskeratosis congenita (DC) is a telomere biology disorder characterized by a mucocutaneous triad, aplastic anemia, and predisposition to cancer. Mutations in a narrow segment of TINF2 exon 6 have been recognized to cause often-severe DC that is either sporadic or autosomal dominant. We describe three children with very early presentations of DC, including one with the severe variant known as Revesz syndrome. Although most TINF2 mutations reported to date are missense changes, each of our patients carried a novel heterozygous nonsense or frameshift mutation, revealing a new 5' boundary to the affected gene segment in patients with DC. Examination of patient-derived lymphoblastoid cell lines revealed stable expression of the predicted truncated TIN2 proteins. In co-immunoprecipitation assays, the ability of a truncation mutant to interact with TRF1 was severely impaired, whereas the ability of the most common DC-associated mutant was much less affected. This suggests that the disruption of TIN2-TRF1 interaction may contribute to the severe clinical phenotype observed in the context of the TIN2 truncation mutation, but is unlikely to be the primary cause of telomere shortening associated with the more prevalent TIN2 missense mutations. Telomere flow-fluorescent in situ hybridization (FISH) analysis of one pedigree showed the dramatic effect a de novo nonsense TINF2 mutation had on telomere length in early development. These cases underscore the severe manifestations of truncating TINF2 mutations.     

Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system

Recent studies have suggested that the use of aminoglycosides to suppress disease-causing nonsense mutations may be a promising new therapy for a large number of genetic diseases. However, gentamicin is currently the only clinically relevant aminoglycoside shown to suppress premature stop mutations in a mammalian system. We compared the ability of the clinically approved aminoglycosides gentamicin, tobramycin, and amikacin to suppress premature stop mutations. Using readthrough reporter constructs as well as mammalian cDNAs containing naturally occurring premature stop mutations, we found that each of these aminoglycosides can suppress many premature stop mutations in a context-dependent manner in a mammalian translation system. Our results indicate that the tetranucleotide termination signal (the stop codon and the nucleotide 3' of the stop codon) is the primary determinant for aminoglycoside-mediated suppression. The levels of termination suppression achieved by tobramycin were substantially lower than those observed with gentamicin. In contrast, amikacin stimulated suppression in a manner that was generally similar to gentamicin. Amikacin produced higher levels of readthrough than gentamicin at some contexts, demonstrating a unique pattern of context dependence. Experiments with mammalian cDNAs confirmed these results and demonstrated that these aminoglycosides can also suppress disease-associated premature stop mutations previously identified in the IDUA gene (responsible for the lysosomal storage disease mucopolysaccharidosis I) and the P53 gene (associated with many forms of cancer). Taken together, these results suggest that amikacin represents an alternative to gentamicin for suppression therapy in certain contexts, thus providing a means of optimizing the efficacy of aminoglycoside-mediated suppression of premature stop mutations.     

Aging syndrome genes and premature coronary artery disease

Background  

Vascular disease is a feature of aging, and coronary vascular events are a major source of morbidity and mortality in rare premature aging syndromes. One such syndrome is caused by mutations in the lamin A/C (LMNA) gene, which also has been implicated in familial insulin resistance. A second gene related to premature aging in man and in murine models is the KLOTHO gene, a hypomorphic variant of which (KL-VS) is significantly more common in the first-degree relatives of patients with premature coronary artery disease (CAD). We evaluated whether common variants at the LMNA or KLOTHO genes are associated with rigorously defined premature CAD.