A Spectrum of Severe Familial Liver Disorders Associate with Telomerase Mutations

Background

Telomerase is an enzyme specialized in maintaining telomere lengths in highly proliferative cells. Loss-of-function mutations cause critical telomere shortening and are associated with the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia and with idiopathic pulmonary fibrosis. Here, we sought to determine the spectrum of clinical manifestations associated with telomerase loss-of-function mutations.

Methodology/Principal Findings

Sixty-nine individuals from five unrelated families with a variety of hematologic, hepatic, and autoimmune disorders were screened for telomerase complex gene mutations; leukocyte telomere length was measured by flow fluorescence in situ hybridization in mutation carriers and some non-carriers; the effects of the identified mutations on telomerase activity were determined; and genetic and clinical data were correlated. In six generations of a large family, a loss-of-function mutation in the telomerase enzyme gene TERT associated with severe telomere shortening and a range of hematologic manifestations, from macrocytosis to acute myeloid leukemia, with severe liver diseases marked by fibrosis and inflammation, and one case of idiopathic pulmonary fibrosis but not with autoimmune disorders. Additionally, we identified four unrelated families in which loss-of-function TERC or TERT gene mutations tracked with marrow failure, pulmonary fibrosis, and a spectrum of liver disorders.

Conclusions/Significance

These results indicate that heterozygous telomerase loss-of-function mutations associate with but are not determinant of a large spectrum of hematologic and liver abnormalities, with the latter sometimes occurring in the absence of marrow failure. Our findings, along with the link between pulmonary fibrosis and telomerase mutations, also suggest a common pathogenic mechanism for fibrotic diseases in which defective telomere repair plays important role.     

Short telomeres, even in the presence of telomerase, limit tissue renewal capacity

Autosomal-dominant dyskeratosis congenita is associated with heterozygous mutations in telomerase. To examine the dosage effect of telomerase, we generated a line of mTR+/- mice on the CAST/EiJ background, which has short telomeres. Interbreeding of heterozygotes resulted in progressive telomere shortening, indicating that limiting telomerase compromises telomere maintenance. In later-generation heterozygotes, we observed a decrease in tissue renewal capacity in the bone marrow, intestines, and testes that resembled defects seen in dyskeratosis congenita patients. The progressive worsening of disease with decreasing telomere length suggests that short telomeres, not telomerase level, cause stem cell failure. Further, wild-type mice derived from the late-generation heterozygous parents, termed wt*, also had short telomeres and displayed a germ cell defect, indicating that telomere length determines these phenotypes. We propose that short telomeres in mice that have normal telomerase levels can cause an occult form of genetic disease.     

Human diseases of telomerase dysfunction: insights into tissue aging

There are at least three human diseases that are associated with germ-line mutations of the genes encoding the two essential components of telomerase, TERT and TERC. Heterozygous mutations of these genes have been described for patients with dyskeratosis congenita, bone marrow failure and idiopathic pulmonary fibrosis. In this review, we will detail the clinical similarities and difference of these diseases and review the molecular phenotypes observed. The spectrum of mutations in TERT and TERC varies for these diseases and may in part explain the clinical differences observed. Environmental insults and genetic modifiers that accelerate telomere shortening and increase cell turnover may exaggerate the effects of telomerase haploinsufficiency, contributing to the variability of age of onset as well as tissue-specific organ pathology. A central still unanswered question is whether telomerase dysfunction and short telomeres are a much more prominent factor than previously suspected in other adult-onset, age-related diseases. Understanding the biological effects of these mutations may ultimately lead to novel treatments for these patients.     

Dyskeratosis congenita: telomerase, telomeres and anticipation

Dyskeratosis congenita (DC) is a rare bone marrow failure syndrome that displays marked clinical and genetic heterogeneity. The identification of dyskeratosis congenita gene 1 (DKC1) mutations in X-linked recessive patients initially suggested that DC is a defective pseudouridylation disorder. The subsequent identification of mutations in the telomerase RNA component (TERC) of autosomal dominant DC patients together with the discovery that both TERC and the DKC1-encoded protein, dyskerin, are closely associated in the telomerase complex have suggested that the pathophysiology of DC predominantly relates to defective telomere maintenance. Recent discoveries have shown that autosomal dominant DC exhibits disease anticipation and that this is associated with progressive telomere shortening owing to the haplo-insufficiency of TERC.     

A role for sister telomere cohesion in telomere elongation by telomerase

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC) a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View, and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.     

A role for sister telomere cohesion in telomere elongation by telomerase

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC), a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and, further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.Key words: telomeres, cohesion, telomerase, TIN2, dyskeratosis congenita     

Stem cells,telomerase and dyskeratosis congenita

Dyskeratosis congenita is a rare skin and bone marrow failure syndrome caused by defective telomere maintenance in stem cells. The major X-linked form of the disease is due to mutations in a nucleolar protein, dyskerin, that is part of small nucleolar ribonucleoprotein particles that are involved in processing ribosomal RNA. It is also found in the telomerase complex, pointing to an unexpected link between these two processes. An autosomal dominant form is due to mutations in the RNA component of telomerase (hTR). Patients with this form of the disease are more severely affected in later generations that carry the mutations, possibly due to the inheritance of shortened telomeres, disguising the inherited nature of the disease in some cases classified as aplastic anemia. Because of the importance of telomerase in tumour formation and aging, study of this disease may provide important clues about these fundamental processes.     

The Shelterin TIN2 Subunit Mediates Recruitment of Telomerase to Telomeres

Dyskeratosis Congenita (DC) is a heritable multi-system disorder caused by abnormally short telomeres. Clinically diagnosed by the mucocutaneous symptoms, DC patients are at high risk for bone marrow failure, pulmonary fibrosis, and multiple types of cancers. We have recapitulated the most common DC-causing mutation in the shelterin component TIN2 by introducing a TIN2-R282H mutation into cultured telomerase-positive human cells via a knock-in approach. The resulting heterozygous TIN2-R282H mutation does not perturb occupancy of other shelterin components on telomeres, result in activation of telomeric DNA damage signaling or exhibit other characteristics indicative of a telomere deprotection defect. Using a novel assay that monitors the frequency and extension rate of telomerase activity at individual telomeres, we show instead that telomerase elongates telomeres at a reduced frequency in TIN2-R282H heterozygous cells; this recruitment defect is further corroborated by examining the effect of this mutation on telomerase-telomere co-localization. These observations suggest a direct role for TIN2 in mediating telomere length through telomerase, separable from its role in telomere protection.     

Dyskeratosis congenita,stem cells and telomeres

Dyskeratosis congenita (DC) is a multi-system disorder which in its classical form is characterised by abnormalities of the skin, nails and mucous membranes. In approximately 80% of cases, it is associated with bone marrow dysfunction. A variety of other abnormalities (including bone, brain, cancer, dental, eye, gastrointestinal, immunological and lung) have also been reported. Although first described almost a century ago it is the last 10 years, following the identification of the first DC gene (DKC1) in 1998, in which there has been rapid progress in its understanding. Six genes have been identified, defects in which cause different genetic subtypes (X-linked recessive, autosomal dominant, autosomal recessive) of DC. The products of these genes encode components that are critical for telomere maintenance; either because they are core constituents of telomerase (dyskerin, TERC, TERT, NOP10 and NHP2) or are part of the shelterin complex that protects the telomeric end (TIN2). These advances have also highlighted the connection between the more “cryptic/atypical” forms of the disease including aplastic anaemia and idiopathic pulmonary fibrosis. Equally, studies on this disease have demonstrated the critical importance of telomeres in human cells (including stem cells) and the severe consequences of their dysfunction. In this context DC and related diseases can now be regarded as disorders of “telomere and stem cell dysfunction”.     

The pathogenesis of COPD and IPF: Distinct horns of the same devil?

New paradigms have been recently proposed in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), evidencing surprising similarities between these deadly diseases, despite their obvious clinical, radiological and pathologic differences. There is growing evidence supporting a "double hit" pathogenic model where in both COPD and IPF the cumulative action of an accelerated senescence of pulmonary parenchyma (determined by either telomere dysfunction and/or a variety of genetic predisposing factors), and the noxious activity of cigarette smoke-induced oxidative damage are able to severely compromise the regenerative potential of two pulmonary precursor cell compartments (alveolar epithelial precursors in IPF, mesenchymal precursor cells in COPD/emphysema). The consequent divergent derangement of signalling pathways involved in lung tissue renewal (mainly Wnt and Notch), can eventually lead to the distinct abnormal tissue remodelling and functional impairment that characterise the alveolar parenchyma in these diseases (irreversible fibrosis and bronchiolar honeycombing in IPF, emphysema and airway chronic inflammation in COPD).     

Telomerase dysfunction and dyskeratosis congenita

Dyskeratosis congenita (DC) is a multi system bone marrow failure syndrome characterized by muco-cutaneous abnormalities and an increased predisposition to malignancy. It exhibits considerable clinical and genetic heterogeneity. X-linked recessive, autosomal dominant and autosomal recessive forms of the disease are recognized. The X-linked recessive form is due to mutations in dyskerin, which is a component of both small nucleolar ribonuclear protein particles and the telomerase complex. Autosomal dominant DC is due to mutations in the RNA component of telomerase, TERC. As dyskerin and TERC are both components of the telomerase complex and all patients with DC have short telomeres it appears that the principal pathology in DC relates to telomerase dysfunction. The gene or genes involved in the recessive form of DC remain elusive, though genes whose products are required for telomere maintenance remain strong candidates. The study of DC has highlighted the critical role of telomerase and the consequences, including premature aging and malignancy, of its dysfunction.     

TIN2 protein dyskeratosis congenita missense mutants are defective in association with telomerase

Dyskeratosis congenita (DC) is a progressive and heterogeneous congenital disorder that affects multiple systems and is characterized by bone marrow failure and a triad of abnormal skin pigmentation, nail dystrophy, and oral leukoplakia. One common feature for all DC patients is abnormally short telomeres and defects in telomere biology. Most of the known DC mutations have been found to affect core components of the telomerase holoenzyme. Recently, multiple mutations in the gene encoding the telomeric protein TIN2 have been identified in DC patients with intact telomerase genes, but the molecular mechanisms underlying TIN2 mutation-mediated DC remain unknown. Here, we demonstrate that ectopic expression of TIN2 with DC missense mutations in human cells led to accelerated telomere shortening, similar to the telomere phenotypes found in DC patients. However, this telomere shortening was not accompanied by changes in total telomerase activity, localization of TIN2, or telomere end protection status. Interestingly, we found TIN2 to participate in the TPP1-dependent recruitment of telomerase activity. Furthermore, DC mutations in TIN2 led to its decreased ability to associate with TERC and telomerase activity. Taken together, our data suggest that TIN2 mutations in DC may compromise the telomere recruitment of telomerase, leading to telomere shortening and the associated pathogenesis.     

Telomerase therapeutics for degenerative diseases

Telomerase is active in early embryonic and fetal development but is down-regulated in all human somatic tissues before birth. Since telomerase is virtually absent or only transiently active in normal somatic cells throughout postnatal life, telomere length gradually decreases as a function of age in most human tissues. Although telomerase repression likely evolved as a tumor suppressor mechanism, a growing body of evidence from epidemiology and genetic studies point to a role of telomerase repression and short telomeres in a broad spectrum of diseases: (a) Humans with shorter than average telomere length are at increased risk of dying from heart disease, stroke, or infection; (b) Patients with Dyskeratosis congenita are born with shortened telomeres due to mutations in telomerase components, suffer from a variety of proliferative tissue disorders, and typically die early of bone marrow failure; and (c) Individuals with long-term chronic stress or infections have accelerated telomere shortening compared to age-matched counterparts. Telomerase activation may prove useful in the treatment of diseases associated with telomere loss. While human cells dividing in culture lose telomeric DNA and undergo changes that mirror certain age- or disease-associated changes in vivo, telomerase transduced cells have extended replicative capacities, increased resistance to stress, improved functional activities in vitro and in vivo, and no loss of differentiation capacity or growth control. In addition, telomerase transduction in vivo can prevent telomere dysfunction and cirrhotic changes in liver of telomerase knockout mice. Thus, pharmacological activation of telomerase has significant potential for the treatment of a broad spectrum of chronic or degenerative diseases.