Philip J. Mason

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.

Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene.

Paroxysmal nocturnal haemoglobinuria (PNH), an acquired clonal blood disorder, is caused by the absence of glycosyl phosphatidylinositol (GPI)‐anchored surface proteins due to a defect in a specific step of GPI‐anchor synthesis. The cDNA of the X‐linked gene, PIG‐A, which encodes a protein required for this step has recently been isolated. We have carried out a molecular and functional analysis of the PIG‐A gene in four cell lines deficient in GPI‐linked proteins, obtained by Epstein‐Barr virus (EBV) transformation of affected B‐lymphocytes from PNH patients. In all four cell lines transfection with PIG‐A cDNA restored normal expression of GPI‐linked proteins. In three of the four cell lines the primary lesion is a frameshift mutation. In two of these there is a reduction in the amount of full‐length mRNA. The fourth cell line contains a missense mutation in PIG‐A. In each case the mutation was present in the affected granulocytes from peripheral blood of the patients, but not in normal sister cell lines from the same patient. These data prove that PNH is caused in most patients by a single mutation in the PIG‐A gene. The nature of the mutation can vary and most likely occurs on the active X‐chromosome in an early haematopoietic stem cell.

Mutations in the genes encoding 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency

In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11β-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11β-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11β-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11β-HSD1 expression and ER NADPH generation with loss of 11β-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS.

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.

Identification of ATPases Pontin and Reptin as Telomerase Components Essential for Holoenzyme Assembly

Telomerase is a multisubunit ribonucleoprotein (RNP) complex that adds telomere repeats to the ends of chromosomes. Three essential telomerase components have been identified thus far: the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC), and the TERC-binding protein dyskerin. Few other proteins are known to be required for human telomerase function, limiting our understanding of both telomerase regulation and mechanisms of telomerase action. Here, we identify the ATPases pontin and reptin as telomerase components through affinity purification of TERT from human cells. Pontin interacts directly with both TERT and dyskerin, and the amount of TERT bound to pontin and reptin peaks in S phase, evidence for cell-cycle-dependent regulation of TERT. Depletion of pontin and reptin markedly impairs telomerase RNP accumulation, indicating an essential role in telomerase assembly. These findings reveal an unanticipated requirement for additional enzymes in telomerase biogenesis and suggest alternative approaches for inhibiting telomerase in cancer.

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.

Unexplained aplastic anaemia, immunodeficiency, and cerebellar hypoplasia (Hoyeraal‐Hreidarsson syndrome) due to mutations in the dyskeratosis congenita gene, DKC1

Hoyeraal‐Hreidarsson (HH) syndrome is a multisystem disorder affecting boys characterized by aplastic anaemia (AA), immunodeficiency, microcephaly, cerebellar‐hypoplasia and growth retardation. Its pathogenesis is unknown. X‐linked dyskeratosis congenita (DC) is an inherited bone‐marrow‐failure syndrome characterized by skin pigmentation, nail dystrophy and leucoplakia which usually develop towards the end of the first decade of life. AA occurs in >90% of cases of DC. We speculated that mutations in the gene responsible for X‐linked DC (DKC1) may account for the HH syndrome, due to the phenotypic similarities between the disease in respect of AA and gender bias. We therefore analysed the DKC1 gene in two HH families. In one family a nucleotide change at position 361(A → G) in exon 5 was found in both affected brothers; in the other family a nucleotide change at position 146(C → T) in exon 3 was found in the affected boys. The finding of these two novel missense DKC1 mutations demonstrates that HH is a severe variant of DC. They also show that mutations in DKC1 can give rise to a very wide clinical spectrum of manifestations. Boys with unexplained AA or immunodeficiency should be tested for mutations in DKC1 even though they may lack diagnostic features of DC.

Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria

Patients with paroxysmal nocturnal haemoglobinuria (PNH) have in their blood two red-cell populations, one normal and one deficient in proteins anchored to the membrane through a glycan phosphatidylinositol (GPI) structure. The PNH abnormality is due to a somatic mutation in the PIG-A gene, whose product is required for an early step in GPI anchor biosynthesis. We show that in two patients, two PNH clones with different mutations co-exist, and must therefore have arisen independently. This finding supports the concept that PNH develops under the pressures of a positive selection mechanism whereby GPI-anchor-deficient haemopoietic cells have a survival advantage.

Dyskeratosis Congenita (DC) Registry: identification of new features of DC

Dyskeratosis congenita (DC) is an inherited disorder characterized by skin pigmentation, nail dystrophy and mucosal leucoplakia. In 1995 a Dyskeratosis Congenita Registry was established at the Hammersmith Hospital. In the 46 families recruited, 76/83 patients were male, suggesting that the major form of DC is X‐linked. As well as a variety of noncutaneous abnormalities, the majority (93%) of patients had bone marrow (BM) failure and this was the principal cause (71%) of early mortality. In addition to BM hypoplasia, some patients also developed myelodysplasia and acute myelod leukaemia. Pulmonary abnormalities were present in 19% of patients. In affected females the phenotype was less severe. Some female carriers of X‐linked DC had clinical features. Carriers of X‐linked DC showed skewed X‐chromosome inactivation patterns (XCIPs), suggesting that cells expressing the normal DC allele have a growth/survival advantage over cells that express the mutant allele. Linkage analysis in multiplex families confirmed that the DKC1 gene, responsible for the X‐linked form of DC, is located within Xq28 and facilitated its positional cloning. The high incidence of BM failure in association with a wide range of somatic abnormalities together with the ubiquitous expression of DKC1 suggest that, as well as having a critical role in normal haemopoiesis, this gene has a key role in normal cell biology.

Dyskeratosis congenita -- a disease of dysfunctional telomere maintenance.

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome associated with abnormalities of the skin, fingernails, and tongue. Other clinical manifestations may include epiphora, lung fibrosis, liver cirrhosis, osteoporosis, and a predisposition to develop a variety of malignancies. The clinical picture often resembles that of a premature aging syndrome and tissues affected are those with a high cell turnover. DC has been linked to mutations in at least four distinct genes, three of which have been identified. The product of these genes, dyskerin, the telomerase RNA (TERC), and the catalytic unit of telomerase (TERT) are part of a ribonucleoprotein complex, the telomerase enzyme, that is essential for the elongation and maintenance of chromosome ends or telomeres. All patients with DC have excessively short telomeres, indicating that the underlying defect in these individuals is an inability to maintain the telomeres. The purpose of the current review is to highlight recent insights into the molecular pathogenesis of DC. We discuss the impact these findings have on our current understanding of telomere function and maintenance, and on the diagnosis, management, and treatment of patients with conditions caused by dysfunctional telomeres.