Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA

Aplastic anaemia in adults is usually acquired, but rarely constitutional types of bone marrow failure can occur late in life. We assessed two families with onset of pancytopenia in adults and detected two novel point mutations in the telomerase RNA gene (TERC) in each family. This gene is abnormal in some kindreds with dyskeratosis congenita. Individuals in our families with mutated TERC did not have physical signs of dyskeratosis congenita, and their blood counts were nearly normal, but all had severely shortened telomeres, reduced haemopoietic function, and raised serum erythropoietin and thrombopoietin. Bone marrow failure of variable severity due to dyskeratosis congenita, historically characterised by associated physical anomalies and early pancytopenia, may be present in otherwise phenotypically normal adults, and can masquerade as acquired aplastic anaemia.     

Heterozygous telomerase RNA mutations found in dyskeratosis congenita and aplastic anemia reduce telomerase activity via haploinsufficiency

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.     

Dyskeratosis congenita: its link to telomerase and aplastic anaemia

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.     

Inherited aplastic anaemia

A number of inherited (constitutional/genetic) disorders are characterized by bone marrow (BM) failure/aplastic anaemia (AA) usually in association with one or more somatic abnormality. Occasionally, these patients may present with AA alone and be labelled to have idiopathic AA. In recent years, there have been significant advances in the genetics of Fanconi anaemia (FA), dyskeratosis congenita (DC) and other BM failure syndromes. This is facilitating accurate diagnosis and beginning to unravel their pathophysiology. Furthermore, these advances are also providing important insights into normal haemopoiesis and how this might become defective in some patients presenting with the more common idiopathic AA. Indeed, a link between DC and idiopathic AA and in turn to defective telomerase has now been established. This advance also suggests that treatments directed at correction of telomerase activity might benefit AA patients who do not respond to conventional therapy.     

Acetazolamide-associated aplastic anaemia

Eleven cases of acetazolamide-associated aplastic anaemia were reported in Sweden during a 17-year period. There were six women and five men with a median age of 71 years (range 63-85 years). The median dose of acetazolamide was 500 mg, and the median duration of treatment was 3 months (range 2-71 months). Ten of the eleven patients died, all within 8 weeks after detection of their aplastic anaemia. The relative risk of developing aplastic anaemia when taking acetazolamide was 13.3 (95% confidence limits (CL); 6.8-25.3). The estimated incidence of reported acetazolamide-associated aplastic anaemia is approximately one in 18,000 patient years. The results strongly indicate that acetazolamide treatment is associated with a substantial increase in the risk of developing aplastic anaemia.     

Mouse dyskerin mutations affect accumulation of telomerase RNA and small nucleolar RNA, telomerase activity, and ribosomal RNA processing

Dyskerin is a nucleolar protein present in small nucleolar ribonucleoprotein particles that modify specific uridine residues of rRNA by converting them to pseudouridine. Dyskerin is also a component of the telomerase complex. Point mutations in the human gene encoding dyskerin cause the skin and bone marrow failure syndrome dyskeratosis congenita (DC). To test the extent to which disruption of pseudouridylation or telomerase activity may contribute to the pathogenesis of DC, we introduced two dyskerin mutations into murine embryonic stem cells. The A353V mutation is the most frequent mutation in patients with X-linked DC, whereas the G402E mutation was identified in a single family. The A353V, but not the G402E, mutation led to severe destabilization of telomerase RNA, a reduction in telomerase activity, and a significant continuous loss of telomere length with increasing numbers of cell divisions during in vitro culture. Both mutations caused a defect in overall pseudouridylation and a small but detectable decrease in the rate of pre-rRNA processing. In addition, both mutant embryonic stem cell lines showed a decrease in the accumulation of a subset of H/ACA small nucleolar RNAs, correlating with a significant decrease in site-specific pseudouridylation efficiency. Interestingly, the H/ACA snoRNAs decreased in the G402E mutant cell line differed from those affected in A353V mutant cells. Hence, our findings show that point mutations in dyskerin may affect both the telomerase and pseudouridylation pathways and the extent to which these functions are altered can vary for different mutations.     

Eosinophilic fasciitis and aplastic anaemia

Aplastic anemia developed in a 66-yr-old farmer with eosinophilic fasciitis. The concurrence of these 2 rare diseases is noteworthy. The case is presented and the relevant literature is briefly reviewed.     

Serum inhibitors in aplastic anaemia

Summary To study the frequency and clinical importance of serum factors inhibiting CFU-c in patients with aplastic anaemia, sera from 21 patients were analysed.The sera of eight patients showed inhibition of at least one normal bone marrow, but strong and consistent inhibition of two or more normal bone-marrow samples was found in only two cases. In both of these two patients the inhibition disappeared after successful therapy. Fair, but not complete, correlation was found between serum hibitors and HLA-antibodies, suggesting that at least part of these inhibitors are antibodies directed against HLA-antigens present on myeloid progenitor cells.Serum inhibitors appear to be infrequent in the population of patients with aplastic anaemia seen in our hospital and appear to have no major implications for therapy or outcome of the disease.     

CFU-C inhibitors in aplastic anaemia

Summary Peripheral blood lymphocytes from 15 patients with marrow aplasia were tested for their ability to inhibit the proliferation of normal granulopoietic precursor cells (CFU-C) in agar culture, relative to the inhibitory effect of normal lymphocytes studied in parallel. Eight of the 15 patients with marrow aplasia had lymphocytes which were significantly less inhibitory to normal CFU-C than controls whereas 3 patients had lymphocytes which were significantly more inhibitory. Two further patients who had recovered from marrow aplasia were also studied. The effect of patient's plasma and normal plasma on normal CFU-C proliferation was also studied and in 1 case a potent inhibitor of granulopoiesis was demonstrated. In 9 cases CFU-C could be cultured from patient's marrow, and parallel studies examining the effects of lymphocytes or plasma on patient's CFU-C. In none of the 9 marrow samples tested was inhibition by patient lymphocytes significantly greater than normal controls.The results highlight the heterogeneity inherent in the study of aplastic states and serve to underline the importance of controls. In only a minority of cases (20%) was lymphocyte suppression of normal granulopoiesis by lymphocytes from patients with aplastic anaemia significantly greater than normal lymphocyte suppression.     

Hypoimmunoglobulinaemia and aplastic anaemia.

Serum immunoglobulin levels were estimated in 19 patients with aplastic anaemia. Although the mean levels for IgG, IgA and IgM for the group as a whole were within the normal range, 8 individual cases showed low levels of one or more immunoglobulin class, suggesting a disturbance in immunoglobulin production. One of these patients had a thymoma, and another positive tests for anti-nuclear factor, but there was no evidence of auto-immune disease in the others. It is suggested that 'aplastic anaemia' may sometimes be a disease due to injury of a stem cell subserving both haemopoietic and immunological function.     

Pathophysiology of aplastic anaemia

It is the conventional opinion that acquired aplastic anaemia is a heterogenous disease including basically different conditions, such as idiopathic or virus induced pancytopenia, toxic-allergic marrow damage or autoimmunity. Here, an alternative concept is proposed, according to which aplastic anaemia is one disease, but multifactorial in all patients, apparent differences being due to the relative prevalence of one or the other pathophysiological component in individual patients. Bone marrow from patients in the severe phase of aplastic anaemia does not grow in culture and is therefore not suitable for experimentation. Alternatively, bone marrow from patients who have resumed some degree of autologous bone marrow function, but still have residual signs of the disease after non-invasive therapy, offers the possibility to study pathophysiological mechanisms in vitro. The majority of experiments presented in this chapter have been done in such patients, assuming that their status of disease in some way reflects the original, more serious pretreatment condition. Three major pathophysiological components will be discussed, and it will be proposed how these factors act in concert to cause or aggravate aplasia.