Anna Marrone

Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC.

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.

Association between aplastic anaemia and mutations in telomerase RNA.

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.

Progress and prospects in rat genetics: a community view

The rat is an important system for modeling human disease. Four years ago, the rich 150-year history of rat research was transformed by the sequencing of the rat genome, ushering in an era of exceptional opportunity for identifying genes and pathways underlying disease phenotypes. Genome-wide association studies in human populations have recently provided a direct approach for finding robust genetic associations in common diseases, but identifying the precise genes and their mechanisms of action remains problematic. In the context of significant progress in rat genomic resources over the past decade, we outline achievements in rat gene discovery to date, show how these findings have been translated to human disease, and document an increasing pace of discovery of new disease genes, pathways and mechanisms. Finally, we present a set of principles that justify continuing and strengthening genetic studies in the rat model, and further development of genomic infrastructure for rat research.

Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita

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.

Defining the Pathogenic Role of Telomerase Mutations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

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 α). 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. Hum Mutat 30:1–7, 2009.

A novel DKC1 mutation, severe combined immunodeficiency (T+B–NK– SCID) and bone marrow transplantation in an infant with Hoyeraal–Hreidarsson syndrome

Summary.  X‐linked Hoyeraal–Hreidarsson syndrome (XL‐HHS) is the severe infantile variant of X‐linked dyskeratosis congenita (XL‐DC) and both are due to mutations in the DKC1 gene within Xq28. We report a novel missense mutation in DKC1 exon 3 (T113→C, Ile38Thr) in a Sardinian infant with XL‐HHS in whom the disease was characterized by ‘T+B–NK–’ severe combined immunodeficiency and bone marrow failure. He underwent sibling bone marrow transplantation using a conditioning regimen (fludarabine, rabbit antithymocyte globulin, low‐dose melphalan) selected according to the HHS/DC phenotype. This was associated with low toxicity, prompt engraftment with adequate immune reconstitution and full donor haemopoiesis.

Dyskeratosis congenita: molecular insights into telomerase function, ageing and cancer

Dyskeratosis congenita (DC) is a severe, inherited, bone marrow failure syndrome, with associated cutaneous and noncutaneous abnormalities. DC patients also show signs of premature ageing and have an increased occurrence of cancer. DC can originate through: (1) mutations in DKC1, which result in X-linked recessive DC; (2) mutations in the RNA component of telomerase (TERC), which result in autosomal dominant DC (AD-DC); and (3) mutations in other, currently uncharacterized, genes, which result in autosomal recessive DC (AR-DC). As DKC1 encodes dyskerin, a protein component of small nucleolar ribonucleoprotein (snoRNP) particles, which are important in ribosomal RNA processing, DC was initially described as a disorder of defective ribosomal biogenesis. Subsequently, dyskerin and TERC were shown to closely associate with each other in the telomerase complex, and DC has since come to be regarded as a telomerase deficiency disorder characterised by shorter telomeres. These findings demonstrate the importance of telomerase in humans and highlight how its deficiency (through DKC1 and TERC mutations) results in multiple abnormalities including premature ageing, bone marrow failure and cancer. Identification of the gene(s) involved in AR-DC will help to define the pathophysiology of DC further, as well as expand our insights into telomere function, ageing and cancer.

Dyskeratosis Congenita: A Disorder of Defective Telomere Maintenance?

Dyskeratosis congenita (DC) is a rare multisystem bone marrow failure syndrome that displays marked clinical and genetic heterogeneity. X-linked recessive, autosomal dominant and autosomal recessive forms of the disease are recognized. The gene that is mutated in the X-linked form of the disease is DKC1. The DKC1 -encoded protein, dyskerin, is a component of small nucleolar ribonucleoprotein particles, which are important in ribosomal RNA processing, and of the telomerase complex. The autosomal dominant form of DC is due to mutations in the gene for the RNA component of telomerase (TERC). Because both dyskerin and TERC are components of the telomerase complex and all patients with DC have short telomeres, the principal pathology of DC appears to relate to telomerase dysfunction, although defects in ribosomal processing via dyskerin’s involvement in pseudouridylation cannot be completely ruled out. The gene or genes involved in autosomal recessive DC remain elusive, although genes whose products are required for telomere maintenance remain strong candidates. The study of DC highlights the importance of telomerase in humans and how its deficiency results in multiple abnormalities, including premature aging, bone marrow failure, and cancer.

A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC) promoter in a patient with Paroxysmal Nocturnal Haemoglobinuria.

BackgroundMutations in the gene coding for the RNA component of telomerase, hTERC, have been found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal blood disorder associated with aplastic anemia and characterized by the presence of one or more clones of blood cells lacking glycosylphosphatidylinositol (GPI) anchored proteins due to a somatic mutation in the PIGA gene.MethodsWe searched for mutations in DNA extracted from PNH patients by amplification of the hTERC gene and denaturing high performance liquid chromatography (dHPLC). After a mutation was found in a potential transcription factor binding site in one patient electrophoretic mobility shift assays were used to detect binding of transcription factors to that site. The effect of the mutation on the function of the promoter was tested by transient transfection constructs in which the promoter is used to drive a reporter gene.ResultsHere we report the finding of a novel promoter mutation (-99C->G) in the hTERC gene in a patient with PNH. The mutation disrupts an Sp1 binding site and destroys its ability to bind Sp1. Transient transfection assays show that mutations in this hTERC site including C-99G cause either up- or down-regulation of promoter activity and suggest that the site regulates core promoter activity in a context dependent manner in cancer cells.ConclusionsThese data are the first report of an hTERC promoter mutation from a patient sample which can modulate core promoter activity in vitro, raising the possibility that the mutation may affect the transcription of the gene in hematopoietic stem cells in vivo, and that dysregulation of telomerase may play a role in the development of bone marrow failure and the evolution of PNH clones.

Dyskeratosis congenita: a disorder of telomerase deficiency and its relationship to other diseases

Dyskeratosis congenita is a heterogeneous inherited bone marrow failure syndrome that is classically characterized by abnormal skin pigmentation, nail dystrophy and leukoplakia. X-linked recessive dyskeratosis congenita is due to mutations in DKC1, which encodes dyskerin. This protein is a key component in pseudouridylation and for telomere maintenance through the stabilization of the telomerase complex. Autosomal dominant dyskeratosis congenita has been found to be due to mutations in TERC and TERT that encode two key components (telomerase RNA component and reverse transcriptase) of the telomerase complex. These observations, together with the finding of shorter than expected telomeres in all dyskeratosis congenita patients, suggest that dyskeratosis congenita is primarily a disorder of telomerase deficiency. At present, it is unclear if defective pseudouridylation and/or rRNA processing plays any role in the pathophysiology of X-linked dyskeratosis congenita, but the clinical phenotype observed in high...