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.     

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.     

Assignment of the gene for dyskeratosis congenita to Xq28

Summary Dyskeratosis congenita is an X-linked recessive disorder with diagnostic dermatological features, bone marrow hypofunction, and a predisposition to neoplasia in early adult life. Linkage analysis was undertaken in an extensive family with the condition using the Xg blood group and 17 cloned X chromosomal DNA sequences which recognise restriction fragment length polymorphisms (RFLPs). No recombination was observed between the locus for dyskeratosis congenita (DKC) and the RFLPs identified by DXS52 (St 14-1) (Z_max=3.33 at _max=0 with 95% confidence limits of 0 to 14 cM). Similarly no recombination was observed for the disease locus and F8 (Z_max=1.23 at _max=0) nor for DXS15 (Z_max=1.62 at _max=0), but both of these markers were only informative in part of the family whereas DXS52 was fully informative. DXS52, DXS15, and F8 are known to be tightly linked and have previously been assigned to Xq28. Thus the gene for dyskeratosis congenita can be assigned to Xq28. These DNA sequence polymorphisms will be of clinical value for carrier detection and prenatal diagnosis.     

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.     

Enhanced G2 chromatid radiosensitivity in dyskeratosis congenita fibroblasts.

Dyskeratosis congenita (DC) is an inherited disorder characterized by reticular pigmentation of the skin, dystrophic nails, mucosal leukoplakia, and a predisposition to cancer in early adult life. In the majority of cases, DC is an X-linked recessive trait. However, one or more autosomal form(s) of DC may exist. Although excessive spontaneous chromatid breakage has been reported in DC, it is not a consistent cytological marker for this disorder. We examined the frequency and specificity of X-irradiation-induced G2 chromatid breakage in fibroblasts from three unrelated DC patients (two males and one female). Metaphase cells from DC patients had significantly more chromatid breaks (16-18-fold and 17-26-fold at 50 and 100 rad X-irradiation, respectively) and chromatid gaps (10-12-fold and 6-7-fold at 50 and 100 rad, respectively) than those from two different controls. Analysis of banded chromosomes revealed a nonrandom distribution of chromatid aberrations in DC but not in controls, a distribution corresponding to some of the known breakpoints for cancer-specific rearrangements, constitutive fragile sites, and/or loci for cellular proto-oncogenes. The significance of this finding for cancer predisposition in DC patients is uncertain, but the increased susceptibility of X-irradiation-induced chromatid breakage may serve as a cellular marker of diagnostic value.     

Skewed X-chromosome inactivation in female carriers of dyskeratosis congenita.

In this study, we report on a family with X-linked dyskeratosis congenita (DC). Linkage analysis with markers in the factor VIII gene at Xq28 yielded a LOD score of 2 at a recombination of 0. Clinical manifestations of DC, such as skin lesions following the Blaschko lines, were present in two obligate carrier females. Highly skewed X inactivation was observed in white blood cells, cultured skin fibroblasts, and buccal mucosa from female carriers of DC in this family. This suggests a critical role for the DC gene in bone marrow-cell and fibroblast-cell proliferation.     

The relationship between DNA methylation and telomere length in dyskeratosis congenita

The regulation of telomere length (TL) is a complex process, requiring the telomerase enzyme complex and numerous regulatory proteins. Epigenetic regulation may also be important in telomere maintenance. Specifically, methylation at subtelomeres is associated with changes in TL in vitro and in mouse models. Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by exceedingly short telomeres and mutations in telomere biology genes. To understand the interaction between methylation and TL in humans, we measured LINE-1, pericentromeric (NBL2), and subtelomeric (D4Z4) methylation in peripheral blood DNA derived from 40 patients with DC and 51 mutation-negative relatives. Pearson's correlation coefficient and linear regression models were used to evaluate the relationship between age-standardized lymphocyte TL measured by flow FISH and % DNA methylation. No differences in % subtelomeric, LINE-1, or pericentromeric methylation between patients with DC and relatives were noted except for an increase in % subtelomeric methylation in DC patients with a telomerase-complex mutation (TERC, TERT, DKC1, or TCAB1) (63.0% in DC vs. 61.8% in relatives, P = 0.03). Positive correlations between TL and DNA methylation at LINE-1 (r = 0.39, P = 0.01) and subtelomeric (r = 0.32, P = 0.05) sites were present in patients with DC. The positive correlation between TL and % LINE-1 methylation was restricted to TINF2 mutations. In contrast, statistically nonsignificant inverse correlations between TL and % LINE-1 (r = -0.17), subtelomeric (r = -0.20) were present in unaffected relatives. This study suggests an interaction between TL and both subtelomeric and LINE-1 methylation, which may be altered based on mutation status of telomere biology genes.     

Telomerase RNA deficiency in peripheral blood mononuclear cells in X-linked dyskeratosis congenita

Compromised renewal and eventual failure of the hematopoietic system in dyskeratosis congenita (DC) have been proposed to arise from a deficiency in telomerase function. Previously, cultured cell lines from patients with X-linked DC were shown to accumulate less telomerase RNA than cell lines from unaffected family members. Here, we report that telomerase RNA deficiency is also present in the circulating lymphocytes of DC patients. We have compared the accumulation levels of telomerase RNA and a panel of other small RNAs in peripheral blood mononuclear cells from an X-linked DC patient and an unaffected maternal carrier and similarly analyzed cultured lymphoblasts from an X-linked DC patient and maternal carrier in a second family. The DC-patient lymphoid cells show a specific defect in telomerase RNA accumulation with or without cell culture. Our findings support the clinical significance of telomerase deficiency and encourage the use of telomerase activation as a disease therapy.     

YNMG tetraloop formation by a dyskeratosis congenita mutation in human telomerase RNA

Autosomal dominant dyskeratosis congenita (DKC) has been linked to mutations in the RNA component of telomerase, the ribonucleoprotein responsible for telomere maintenance. Recent studies have investigated the role of the GC (107-108) --> AG mutation in the conserved P3 helix in the pseudoknot domain of human telomerase RNA. The mutation was found to significantly destabilize the pseudoknot conformation, resulting in a shift in the thermodynamic equilibrium to favor formation of a P2b hairpin intermediate. In the wild-type sequence, the hairpin intermediate was found to form a novel sequence of pyrimidine base pairs in a continuous stem capped by a structured pentaloop. The DKC mutant hairpin was observed to be slightly more stable than the wild-type hairpin, further shifting the pseudoknot-hairpin equilibrium to favor the mutant P2b hairpin. Here we examined the solution structure of the DKC mutant hairpin to identify the reason for this additional stability. We found that the mutant hairpin forms the same stem structure as wild-type and that the additional stabilization observed using optical melting can be explained by the formation of a YNMG-type tetraloop structure, with the last nucleotide of the pentaloop bulged out into the major groove. Our results provide a structural explanation for the increased stability of the mutant hairpin and further our understanding of the effect of this mutation on the structure and stability of the dominant conformation of the pseudoknot domain in this type of DKC.     

Bleomycin hypersensitivity in dyskeratosis congenita fibroblasts, lymphocytes, and transformed lymphoblasts

We studied the responses of several dyskeratosis congenita (DC) cell lines to the DNA strand-cleaving and base-damaging agent bleomycin. Fibroblasts, peripheral blood lymphocytes, and transformed lymphoblasts of six DC patients and an obligate DC heterozygote showed more chromatid breaks than did respective controls exposed to various concentrations of bleomycin during the G2 phase of the cell cycle (P less than 0.0001). Unsynchronized DC fibroblasts in culture also showed decreased survival, compared to normals, following bleomycin treatment. DC lymphocytes treated with bleomycin for the final 24 h of culture showed more chromatid- and chromosome-type damage than did normals (P less than 0.0001) or G0-treated DC lymphocytes. Spontaneous chromosome breakage was normal in all six DC cell lines. The ability to distinguish affected and heterozygous DC cells without spontaneous chromosome instability from normals on the basis of their bleomycin hypersensitivity provides a marker for future studies of the pathogenesis of this disorder.     

Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita

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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.     

X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene.

Dyskeratosis congenita is a rare inherited bone marrow-failure syndrome characterized by abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia. More than 80% of patients develop bone-marrow failure, and this is the major cause of premature death. The X-linked form of the disease (MIM 305000) has been shown to be caused by mutations in the DKC1 gene. The gene encodes a 514-amino-acid protein, dyskerin, that is homologous to Saccharomyces cerevisiae Cbf5p and rat Nap57 proteins. By analogy to the homologues in other species, dyskerin is predicted to be a nucleolar protein with a role in both the biogenesis of ribosomes and, in particular, the pseudouridylation of rRNA precursors. We have determined the genomic structure of the DKC1 gene; it consists of 15 exons spanning a region of 15 kb. This has enabled us to screen for mutations in the genomic DNA, by using SSCP analysis. Mutations were detected in 21 of 37 additional families with dyskeratosis congenita that were analyzed. These mutations consisted of 11 different single-nucleotide substitutions, which resulted in 10 missense mutations and 1 putative splicing mutation within an intron. The missense change A353V was observed in 10 different families and was shown to be a recurring de novo event. Two polymorphisms were also detected, one of which resulted in the insertion of an additional lysine in the carboxy-terminal polylysine domain. It is apparent that X-linked dyskeratosis congenita is predominantly caused by missense mutations; the precise effect on the function of dyskerin remains to be determined.