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The aim of this study is to present the limbal stem cell deficiency (LSCD) cases with features resembling dyskeratosis congenita (DC), a heritable disease of stem cells principally caused by telomerase deficiency. The clinical, laboratory and molecular findings of four cases are presented. A complete systemic examination was performed in a standardized manner for each patient. Laboratory measurements included investigations of the tests used for screening DC. All eight known disease-causing genes in DC (DKC1, TERC, TERT, NOP10, NHP2, TINF2, C16orf57, and TCAB1) were screened for mutations. The family members of the cases were also assessed, when possible. In all four patients, multisystem involvement was present, along with the disorder affecting corneal LSCs. The affected tissues were mainly the skin and its adnexa, the oral cavity and the hematopoietic system, which are rapidly renewing tissues, consistent with the presence of a stem cell disorder. Similarly affected cases were seen in different generations in families, suggesting an underlying inherited disorder. No mutation was detected in any of the known disease-causing genes in these patients. Based on the presented cases and with the contribution of the review of previously reported DC cases available, we suggest that DC is one of the inherited causes of LSCD and that those cases presenting with LSCD might represent a subgroup of DC caused by mutations in an as yet undefined gene.

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., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a dyskerin.hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with dyskerin. We then investigated mutated forms of hTR and dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the dyskerin.hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin.hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin.hTR interaction may contribute to X-linked DC.

The primary pathology in many cases of myelodysplasia (MDS) and acute myeloid leukemia (AML) remains unknown. In some cases, two or more affected members have been identified in the same family. To date, mutations in two genes have been directly implicated: the hematopoietic transcription factors RUNX1 (runt-related transcription factor 1) and CEBPA (CCATT-box enhancer binding protein alpha). However, there are also other familial cases of MDS/AML where the genetic basis remains unknown. Both MDS, and to a lesser extent AML, have been observed in cases of the bone marrow failure syndrome dyskeratosis congenita, in which telomerase mutations have been identified. Recently, an increased incidence of telomerase reverse transcriptase mutations has been reported in a series of de novo AML. We have now identified novel mutations in the telomerase RNA (TERC) or telomerase reverse transcriptase component (TERT) within 4 of 20 families presenting with familial MDS/AML. Functional analysis has demonstrated that all mutations adversely impact on telomerase activity in vitro, and affected individuals have short telomeres. These families, in conjunction with a review of previously published cases, help to further define the pathological role of telomerase mutations in MDS/AML and have implications for the biology, treatment and screening regimen of de novo cases.

Dyskeratosis congenita (DC) is an inherited multi-system disorder characterised by muco-cutaneous abnormalities, bone marrow failure and a predisposition to malignancy. Bone marrow failure is the principal cause of mortality and is thought to be the result of premature cell death in the haematopoietic compartment because DC cells age prematurely and tend to have short telomeres. DC is genetically heterogeneous and patients have mutations in genes that encode components of the telomerase complex (DKC1, TERC, TERT, NOP10 and NHP2), and telomere shelterin complex (TINF2), both important in telomere maintenance. Here, we transduced primary T lymphocytes and B lymphocyte lines established from patients with TERC and DKC1 mutations with wild type TERC-bearing lentiviral vectors. We found that transduction with exogenous TERC alone was capable of increasing telomerase activity in mutant T lymphocytes and B lymphocyte lines and improved the survival and thus overall growth of B-lymphocyte lines over a prolonged period, regardless of their disease mutation. Telomeres in TERC-treated lines were longer than in the untreated cultures. This is the first study of its kind in DC lymphocytes and the first to demonstrate that transduction with TERC alone can improve cell survival and telomere length without the need for exogenous TERT.

Dyskeratosis congenita is a premature aging syndrome characterized by muco-cutaneous features and a range of other abnormalities, including early greying, dental loss, osteoporosis, and malignancy. Dyskeratosis congenita cells age prematurely and have very short telomeres. Patients have mutations in genes that encode components of the telomerase complex (dyskerin, TERC, TERT, and NOP10), important in the maintenance of telomeres. Many dyskeratosis congenita patients remain uncharacterized. Here, we describe the analysis of two other proteins, NHP2 and GAR1, that together with dyskerin and NOP10 are key components of telomerase and small nucleolar ribonucleoprotein (snoRNP) complexes. We have identified previously uncharacterized NHP2 mutations that can cause autosomal recessive dyskeratosis congenita but have not found any GAR1 mutations. Patients with NHP2 mutations, in common with patients bearing dyskerin and NOP10 mutations had short telomeres and low TERC levels. SiRNA-mediated knockdown of NHP2 in human cells led to low TERC levels, but this reduction was not observed after GAR1 knockdown. These findings suggest that, in human cells, GAR1 has a different impact on the accumulation of TERC compared with dyskerin, NOP10, and NHP2. Most of the mutations so far identified in patients with classical dyskeratosis congenita impact either directly or indirectly on the stability of RNAs. In keeping with this effect, patients with dyskerin, NOP10, and now NHP2 mutations have all been shown to have low levels of telomerase RNA in their peripheral blood, providing direct evidence of their role in telomere maintenance in humans.

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and an increased predisposition to malignancy. X-linked DC is due to mutations in DKC1, while heterozygous mutations in TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) have been found in autosomal dominant DC. Many patients with DC remain uncharacterized, particularly families displaying autosomal recessive (AR) inheritance. We have now identified novel homozygous TERT mutations in 2 unrelated consanguineous families, where the index cases presented with classical DC or the more severe variant, Hoyeraal-Hreidarsson (HH) syndrome. These TERT mutations resulted in reduced telomerase activity and extremely short telomeres. As these mutations are homozygous, these patients are predicted to have significantly reduced telomerase activity in vivo. Interestingly, in contrast to patients with heterozygous TERT mutations or hemizygous DKC1 mutations, these 2 homozygous TERT patients were observed to have higher-than-expected TERC levels compared with controls. Collectively, the findings from this study demonstrate that homozygous TERT mutations, resulting in a pure but severe telomerase deficiency, produce a phenotype of classical AR-DC and its severe variant, the HH syndrome.

Background and Objectives: Functional characterization of heterozygous TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) mutations found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia (AA) shows that telomerase function is defective and that this is associated with short telomeres. This leads to reduced cell longevity with maximal impact on tissues with high proliferate potential. The aim of this study was to establish the role of TERC in the pathophysiology of uncharacterized patients with AA with some features of DC., Design and Methods: The TERC gene was screened for mutations by denaturing high performance liquid chromatography. To determine the functional significance of TERC mutations telomerase activity was assessed in an in vitro (TRAP) assay and telomere length of patients' samples was determined using Southern blot analysis. RESULTS This study led to the identification of four novel TERC mutations (G178A, C180T, D52-86 and G2C) and a recurrent TERC mutation (D110-113GACT)., Interpretation and Conclusions: Two of the de novo TERC mutations (G178A and C180T) found uniquely produce a clinical phenotype in the first generation, differing from previously published cases in which individuals in the first generation are usually asymptomatic. Curiously these mutations are located near the triple-helix domain of TERC. We also observed that the recurrent D110-113GACT can present with AA, myelodysplasia or leukemia. The D52-86 is associated with varied phenotypes including pulmonary disease (pulmonary fibrosis) as the first presentation. In summary, this study reports the functional characterization of several novel TERC mutations associated with varied hematologic and extra-hematologic presentations.

Dyskeratosis congenita (DC) is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. It exhibits marked clinical and genetic heterogeneity. DKC1 encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein (snoRNP) particles is mutated in X-linked recessive DC. Telomerase RNA component (TERC), the RNA component and TERT the enzymatic component of telomerase, are mutated in autosomal dominant DC, suggesting that DC is primarily a disease of defective telomere maintenance. The gene(s) involved in autosomal recessive DC remains elusive. This paper describes studies aimed at defining the genetic basis of AR-DC. Homozygosity mapping in 16 consanguineous families with 25 affected individuals demonstrates that there is no single genetic locus for AR-DC. However, we show that NOP10, a component of H/ACA snoRNP complexes including telomerase is mutated in a large consanguineous family with classical DC. Affected homozygous individuals have significant telomere shortening and reduced TERC levels. While a reduction of TERC levels is not a universal feature of DC, it can be brought about through a reduction of NOP10 transcripts, as demonstrated by siRNA interference studies. A similar reduction in TERC levels is also seen when the mutant NOP10 is expressed in HeLa cells. These findings identify the genetic basis of one subtype of AR-DC being due to the first documented mutations in NOP10. This further strengthens the model that defective telomere maintenance is the primary pathology in DC and substantiates the evidence in humans for the involvement of NOP10 in the telomerase complex and telomere maintenance.

Mutations in TERC, encoding the RNA component of telomerase, have been found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia (AA). Several polymorphisms also exist in the TERC gene, making functional testing of potential pathogenic mutations essential. Here, we have tested normal and mutant TERC molecules in 2 telomerase reconstitution assays, 1 in vitro and 1 in transfected telomerase-negative cells. We find that 2 polymorphic mutations G58A and G228A have no effect on telomerase activity in these assays, whereas 6 mutations found in DC and AA cause reduction or abolition of telomerase activity. Mutations in the pseudoknot region of the TERC molecule, C72G, 96-7DeltaCT, GC107-8AG and 110-3DeltaGACT reduce the catalytic activity of reconstituted telomerase, whereas mutations in the 3' portion of the molecule C408G and a deletion of the 3' 74 bases have normal activity in vitro but reduced intracellular activity. By analyzing second site mutations that recreate regions of secondary structure but retain the pathogenic mutations we show that mutations C72G, GC107-8AG, and C408G act by disrupting the secondary structure or folding of TERC. Finally, experiments reconstituting telomerase with both normal and mutant TERC molecules suggest the mutations act via haploinsufficiency rather than by a dominant-negative mechanism.

Telomerase is a ribonucleoprotein complex that is required to synthesize DNA repeats at the ends of each chromosome. The RNA component of this reverse transcriptase is mutated in the bone marrow failure syndrome autosomal dominant dyskeratosis congenita. Here we show that disease anticipation is observed in families with this disease and that this is associated with progressive telomere shortening.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome exhibiting considerable clinical and genetic heterogeneity. X-linked recessive, autosomal dominant and autosomal recessive forms are recognised. The gene mutated in X-linked DC (DKC1) encodes a highly conserved nucleolar protein called dyskerin. Dyskerin associates with the H/ACA class of small nucleolar RNAs which are important in guiding the conversion of uracil to pseudouracil in ribosomal RNA. Dyskerin also associates with the RNA component of telomerase (hTR) which is important in the maintenance of telomeres. Mutations in hTR were recently demonstrated in patients with autosomal dominant DC and in a subset of patients with aplastic anaemia (AA) but without other diagnostic features of DC. This discovery demonstrates that both DC and a subset of AA are due to a defect in telomerase. The link between DC and AA and in turn to defective telomerase suggests that treatments directed at correction of telomerase activity might benefit DC/AA patients who do not respond to conventional therapy.

The main cause of aplastic anaemia remains elusive. Germline mutations in the gene encoding the RNA component of telomerase (hTR) have been seen in the autosomal dominant form of dyskeratosis congenita--an inherited syndrome characterised by aplastic anaemia. By screening the hTR gene, we identified mutations in two of 17 patients with idiopathic aplastic anaemia, three of 27 patients with constitutional aplastic anaemia, but in none of 214 normal controls (p<0.0001). Furthermore, patients with hTR mutations had significantly shorter telomeres than age-matched controls (p=0.027). These data indicate that, in a subset of patients with aplastic anaemia, the disorder might be associated with a genetic lesion in the telomere maintenance pathway.

Dyskeratosis congenita is a progressive bone-marrow failure syndrome that is characterized by abnormal skin pigmentation, leukoplakia and nail dystrophy. X-linked, autosomal recessive and autosomal dominant inheritance have been found in different pedigrees. The X-linked form of the disease is due to mutations in the gene DKC1 in band 2, sub-band 8 of the long arm of the X chromosome (ref. 3). The affected protein, dyskerin, is a nucleolar protein that is found associated with the H/ACA class of small nucleolar RNAs and is involved in pseudo-uridylation of specific residues of ribosomal RNA. Dyskerin is also associated with telomerase RNA (hTR), which contains a H/ACA consensus sequence. Here we map the gene responsible for dyskeratosis congenita in a large pedigree with autosomal dominant inheritance. Affected members of this family have an 821-base-pair deletion on chromosome 3q that removes the 3' 74 bases of hTR. Mutations in hTR were found in two other families with autosomal dominant dyskeratosis congenita.