Dyskeratosis congenita in all its forms

Authors


Dr Inderjeet Dokal, Department of Haematology, Imperial College School of Medicine (ICSM), Hammersmith Campus, Du Cane Road, London W2 0NN, UK. E-mail: i.dokal@ic.ac.uk

Classic dyskeratosis congenita (DC) is an inherited disease characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leucoplakia ( Zinsser, 1906; Engman, 1926; Cole et al, 1930 ). A variety of non-cutaneous (dental, gastrointestinal, genitourinary, neurological, ophthalmic, pulmonary and skeletal) abnormalities have also been reported ( Sirinavin & Trowbridge, 1975; Drachtman & Alter, 1995; Dokal, 1996a; Knight et al, 1998a ). Bone marrow (BM) failure is the principal cause of early mortality with an additional predisposition to malignancy and fatal pulmonary complications. X-linked recessive, autosomal dominant and autosomal recessive forms of the disease are recognized.

Since the annotation ( Dokal, 1996a), there have been significant advances in DC. These have been facilitated by the dyskeratosis congenita registry (DCR), established at the Hammersmith Hospital (London) in 1995. By November 1999, 92 DC families (Argentina, one; Australia, two; Austria, one; Belgium, two; Brazil, eight; Canada, two; Egypt, one; France, 11, Germany, three; Holland, two; Hong Kong, two; India, two; Ireland, five; Italy, four; New Zealand, one; Spain, two; Turkey, three; United Arab Emirates, two; UK, 14; and USA, 24) had been recruited. These 92 families from 20 different countries collectively comprised 148 (127 male and 21 female) patients. As well as confirming previous observations, the DCR has identified new features of DC and has been pivotal in the identification of the DKC1 gene which is mutated in X-linked DC.

Clinical aspects

In 76 out of the 92 families, there were only males affected and these collectively comprised 118 patients. In 25 of these 76 families, there were two or more affected males with lack of male-to-male transmission, consistent with an X-linked recessive pattern of inheritance. In the 51 families with affected sporadic males, it is likely that many of these also represent the X-linked form of the disease, although some may represent autosomal forms of DC. Overall, out of the 148 patients, 127 (86%) were male and this confirms previous reports that the major form of DC is X linked.

In 16 families out of the total of 92, there was one or more affected female. In 4 of these 16, the cases were sporadic with a history of parental consanguinity in two; in eight families, there were two affected members in the same generation with a history of parental consanguinity in two; in three families, there were affected cases in two different generations; finally, in one out of the 16 families, the two cases were first maternal cousins. Thus, collectively they are likely to represent different genetic subtypes. Some of these are likely to represent autosomal recessive forms of the disease and others autosomal dominant. These families therefore provide further evidence for autosomal recessive and dominant forms of the disease in addition to those published previously ( Sorrow & Hitch, 1963; Sirinavin & Trowbridge, 1975; Tchou & Kohn, 1982; Ling et al, 1985 ; Juneja et al, 1987 ; Pai et al, 1989a ; Drachtman & Alter, 1992; Joshi et al, 1994 ; Knight et al, 1998a ; Elliott et al, 1999 ).

Families with affected males only

Somatic abnormalities A wide range of somatic abnormalities were seen as listed in Table I. DC may therefore be regarded as an inherited multisystem syndrome. In general, the abnormalities were not neonatal in manifestation, but developed progressively at a variable rate. The mucocutaneous features (skin pigmentation, nail dystrophy and leucoplakia; Fig 1) usually appeared between the ages of 5 and 10 years. The median ages of onset for abnormal skin pigmentation, nail dystrophy and leucoplakia were 8 years (range 0·5–21 years), 6 years (range 1–17 years) and 7 years (range 1–26 years) respectively. There was a wide age range over which these features developed and there were also significant qualitative differences in the skin pigmentation and the nail dystrophy; for example, some patients had a very florid rash involving most of the skin, others only had a localized rash and some patients had minimal nail changes, whereas some developed complete nail loss. In families with two or more affected members, the phenotypes among the different individuals tended to be similar. However, in some instances there was significant variability in the severity of the clinical phenotype in different members of the same family. This suggests that the phenotype may be modified by other genetic and/or environmental factors.

Figure 1.

Photographs of DC patients showing abnormal skin pigmentation (A, B, C and D), nail dystrophy of finger nails (E) and toe nails (F and G) and leucoplakia of tongue (H).

A subset (20·3%) of patients developed pulmonary complications (Table I) with reduced diffusion capacity and/or restrictive defect. It has been possible to study some of these patients at several time points. For example, in one patient (aged 36 years) the diffusion capacity (TLCO) fell from 73% to 59% over a period of 6 months, suggesting that pulmonary abnormalities also progress with age and highlighting the need for regular monitoring. Postmortem studies on two patients who died suddenly from acute respiratory disease showed abnormal levels of pulmonary fibrosis and abnormalities in the pulmonary microvasculature. These histological changes correlate with the abnormalities in fibroblasts ( Scappaticci et al, 1989 ; Dokal et al, 1992 ; Kehrer & Krone, 1992; Kehrer et al, 1992 ) observed in DC skin biopsies and the telangiectatic vessels seen at the skin surface clinically. The development of pulmonary abnormalities highlighted by the DCR and previous reports ( Paul et al, 1992 ; Verra et al, 1992 ) may in part explain the high incidence of early and late fatal pulmonary complications after bone marrow transplantation (BMT) ( Berthou et al, 1991 ; Dokal et al, 1992 ; Langston et al, 1996 ; Yabe et al, 1997 ; Rocha et al, 1998 ).

Haematological abnormalities Bone marrow failure resulting in peripheral cytopenias appears to be much more frequent than previously thought. As can be seen from Table II, 85·5% of patients had a peripheral cytopenia of one or more lineages, with 76·3% having a cytopenia of two or more lineages; in 80% of the patients who developed pancytopenia, the age of onset was less than 20 years (median 8 years), with 50% developing pancytopenia below the age of 10 years. Accepting that there may be some bias in the patients referred to the DCR, the actual probability of developing BM failure [one or more peripheral cytopenia(s)] is much higher than previously documented, approaching 94% by the age of 40 years ( Fig 2). This is also reflected in the causes of death (see below). It is noteworthy that one patient (aged 29 years) had approximately 10% myeloid blasts in the BM ( Dokal et al, 1992 ) and three others had hypocellular marrows with features of myelodysplasia (MDS). Thus, like Fanconi's anaemia (FA) ( Auerbach et al, 1998 ), although hypoplasia is the main abnormality seen in the BM there is a predisposition to both MDS and acute myeloid leukaemia (AML) in patients with DC.

Table II.   Haematological complications in DC families with affected males only.
Haematological abnormality% of patients
  1. Median age for onset of pancytopenia was 10 years.

Pancytopenia76·3
Thrombocytopenia only6·6
Leucopenia only2·6
No cytopenia14·5
Figure 2.

Probability of bone marrow failure in dyskeratosis congenita.

Families with affected females

The 16 families (DCR014, DCR0I9, DCR022, DCR024, DCR028, DCR039, DCR049, DCR063, DCR070, DCR073, DCR079, DCR082, DCR083, DCR086, DCR087 and DCR088) with affected females collectively comprised 21 female and nine male cases. Several of the men had features that were similar to those seen in families with affected males only (see above). Indeed, in two families (DCR019 and DCR024), the diagnosis of DC in the female members was facilitated by the diagnosis of the affected male patient.

The phenotype in the female cases varied considerably. In family DCR014, the diagnosis in the case of the sporadic female case was based on the presence of the mucocutaneous triad associated with aplasia below the age of 10 years. In family DCR019, the female case was the daughter of a male patient. She developed BM failure at the age of 4 years without cutaneous features. In family DCR022, both females developed BM failure, they had fewer than two nails affected and had no skin pigmentation. In family DCR024, the female case died of BM failure aged 8 years; her brother was alive and had all the classic features seen in the male patients (see above). In family DCR028, the patient had skin pigmentation abnormalities, leucoplakia, thrombocytopenia and carcinoma of the larynx, but she had no nail dystrophy at the age of 46 years. In family DCR039 (previously published by Elliott et al, 1999 ), the male patient died from BM failure aged 8 years; his sister had mild skin pigmentation, leucoplakia and nail dystrophy. The parents of these two siblings were first cousins. In family DCR063, the 12-year-old girl had reticulate skin pigmentation, leukoplakia and hypocellular BM but no nail dystrophy; her 10-year-old brother had nail dystrophy as well as all the other features present in his older sister. In family DCR070, there were two affected sisters and one affected brother in one generation and their parents were first cousins. In family DCR073, the affected 3-year-old girl had skin pigmentation, hyperconvexed nails, dental caries, intracranial calcification, large cistern magna, liver dysfunction and hypocellular/dysplastic marrow. Her parents were first cousins. DCR079 was an Irish family in which there were two affected sisters with early onset of BM failure (associated with leucoplakia/tongue ulceration, short stature and ataxia), one of whom died from sepsis aged 2 years. In family DCR082, the affected members were a mother and daughter; the daughter had skin pigmentation and leucoplakia but no nail dystrophy, whereas the mother (aged 52 years) had skin pigmentation, macrocytic anaemia and a low CD4 count that had been associated with Pneumocystis carinii pneumonia. These two women therefore had mild disease which appeared to be segregating as an autosomal dominant trait. In family DCR083, there were three affected members in two generations, a sister and brother and their father. The sister died aged 30 years from respiratory complications; she had skin pigmentation, pancytopenia and squamous carcinoma of the tongue. Her brother, aged 47 years, had aplastic anaemia, severe acne and absent fingerprints. Their father was transfusion dependent and is said to have died from iron overload. The three members in this family had mild mucocutaneous features and appeared to show autosomal dominant transmission of the clinical phenotype. In family DCR086, there was one affected girl aged 2 years and her parents were first cousins. She had severe intrauterine growth retardation, microcephaly, developmental delay, ataxia, leucoplakia and severe BM failure at age 2 years. In DCR087, there were three affected members in one sibship, two sisters and their brother. They all had peripheral cytopenia, the brother also had significant atrioventricular (AV) malformation. In family DCR088, the two affected members were first cousins. The index case was a boy who presented at age 4 years with BM failure. He subsequently developed reticulate skin pigmentation, thin hair and extensive dental caries. At the age of 18 years, he still had no nail dystrophy and no leucoplakia. His maternal cousin had aplastic anaemia which responded to steroids and she had very thin hair.

It can be seen that the clinical features in these families with female cases are very variable. In general, in families in which the inheritance appears to be autosomal recessive (e.g. DCR022, DCR024, DCR039, DCR 070, DCR079 and DCR086), the phenotype is more severe than in those in which it appears to be autosomal dominant (e.g. DCR019, DCR082 and DCR083). This agrees broadly with previous literature (autosomal recessive: Sorrow & Hitch, 1963; Ling et al, 1985 ; Juneja et al, 1987 ; Pai et al, 1989a ; Drachtman & Alter, 1992; Joshi et al, 1994 ; Elliott et al, 1999 ; autosomal dominant: Tchou & Kohn, 1982; Gasparini et al, 1985 ) and provides further evidence for autosomal recessive and dominant forms of DC.

Causes of death

Analysis from the DCR shows that 67% of deaths resulted directly from BM failure or from complications of its treatment. Nine per cent died from sudden pulmonary complications. In a further 9%, fatal pulmonary complications were seen in the context of a BMT. Six per cent died from malignancy and 9% died of causes unrelated to DC and its treatment (rabies, myocardial infarction, road traffic accident). The majority of deaths were due to infection and these usually occurred (∼ 80%) before the end of the second decade of life. It is becoming increasingly recognized from patients on the DCR and from the previous reports that immunological abnormalities (including reduced or elevated immunoglobulin levels, reduced B- and/or T-lymphocyte numbers and reduced or absent responses to phytohaemagglutinin) can occur in a subgroup of patients with or without associated BM hypoplasia. Some deaths from infection may thus be attributable to immunodeficiency rather than aplastic anaemia. Fatal opportunistic infections such as Pneumocystis carinii pneumonia and cytomegalovirus have been previously reported ( Wiedemann et al, 1984 ; Rose & Kern, 1992) and this aspect, with special consideration of immunological abnormalities (see Table III), was recently reviewed by Solder et al (1998) .

Malignancy

Malignancies developed in 13 (8·8%) out of the 148 patients. These included four cases of myelodysplasia (two patients with refractory anaemia and two patients with refractory anaemia with excess blasts), one which developed at age 10, one at 22, one at 27 and one at 29 years. There was also one case of Hodgkin's lymphoma (25 years) and eight cases of carcinoma: bronchus (56 years), colon (20 years), larynx (47 years), oesophagus (38 years), pancreas (29 years), skin (20 years) and tongue (one at 30 and one at 38 years). Of the 13 malignancies, three were in female patients (one case of MDS and two cases of carcinoma) and the majority of malignancies developed in the third decade or later.

Current treatments for the bone marrow failure

Treatment for severe BM failure in DC remains unsatisfactory. As in Fanconi's anaemia, the anabolic steroid oxymetholone can produce an improvement in haemopoietic function in many patients for a variable period of time ( Smith et al, 1979 ; unpublished observations). Transient successful responses to granulocyte–macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and erythropoietin have also been reported ( Russo et al, 1990 ; Alter et al, 1997 ). The main treatment for severe aplastic anaemia (SAA), however, is allogeneic stem cell transplantation (SCT) and there is some experience of using both sibling and alternative stem cell donors. To date, SCT has been performed in eight of the patients on the registry and some of these details have been published previously ( Berthou et al, 1991 ; Dokal et al, 1992 ; Forni et al, 1993 ; Knight et al, 1998a ; Rocha et al, 1998 ; Lau et al, 1999 ). Four are currently alive, three of whom had sibling donors (one now at 4, one at 5 and one at 7 years after SCT) and the fourth had an unrelated SCT 3 years ago. There are also other reports of the use of SCT in patients with DC with some long-term survivors ( Philips et al, 1992 ; Langston et al, 1996 ; Yabe et al, 1997 ; Ghavamzadeh et al, 1999 ). Unfortunately, because of early and late fatal pulmonary/vascular complications after SCT ( Berthou et al, 1991 ; Dokal et al, 1992 ; Langston et al, 1996 ; Yabe et al, 1997 ; Knight et al, 1998a; Rocha et al, 1998 ), the results of allogeneic SCT have been less successful than in FA. The presence of pulmonary disease in a significant proportion of DC patients (Table I) perhaps now explains the high incidence of fatal pulmonary complications in the setting of SCT. It also highlights the need to avoid drugs which are associated with pulmonary toxicity (such as busulphan) and to use pulmonary shielding if radiotherapy is used for SCT. As BM failure is the main cause of premature death in DC patients and SCT is currently the only curative option for the BM failure (with some long-term survivors), SCT should continue to be performed on carefully selected patients. Perhaps the best candidates for SCT are patients with no pre-existing pulmonary disease and who have sibling donors. SCT using fludarabine-based protocols appears to be giving encouraging results in FA patients and may be worth exploring in patients with DC. There is a great need to develop new and better treatment strategies for DC patients with SAA.

Dc cell phenotype

Haemopoietic progenitors

Haemopoietic progenitor studies ( Colvin et al, 1984 ; Friedland et al, 1985 ; Alter et al, 1992; Dokal et al, 1992 ; Marsh et al, 1992 ) have shown reduced numbers of all progenitors (erythroid, myeloid and megakaryocytic) compared with controls and there is usually a downward decline with time. The degree to which the progenitors are reduced can vary from patient to patient and they can be reduced even when the PB count is normal (unpublished observations). The haemopoietic system appears to undergo ‘premature ageing’ with a reduction in the proliferative potential of clonogenic progenitors and a reduced subplating cloning efficiency ( Marley et al, 1999 ; unpublished data). The demonstration of abnormalities of growth and chromosomal rearrangements in fibroblasts suggests that the BM failure is likely to be a consequence of abnormalities in both haemopoietic stem cells ( Friedland et al, 1985 ; Marsh et al, 1992 ) and stromal cells.

Chromosomal instability

DC has many features in common with Fanconi's anaemia in which there is a hypersensitivity to clastogenic agents such as mitomycin C (MMC), which is a DNA alkylator. Some authors have reported excessive chromosome breakage in DC, either spontaneous ( Morrison, 1974) or induced ( Pai et al, 1989b ; DeBauche et al, 1990 ; Ning et al, 1992 ), whereas others disputed their findings ( Sirinavin & Trowbridge, 1975; Drachtman & Alter, 1992) with the result that, until recently, confusion existed over the susceptibility of DC cells to clastogenic agents. Coulthard et al (1998) chose a range of agents, based on previous reports suggesting susceptibility, to determine which agents would produce an increased level of breakage in DC lymphocytes over that seen in normal controls. This study demonstrated that there was no significant difference in chromosomal breakage between DC and normal lymphocytes with or without the use of bleomycin, diepoxybutane (DEB), MMC and γ-irradiation. This conclusive study now enables DC patients to be distinguished from FA.

Primary DC skin fibroblasts are abnormal both in morphology and in growth rate. Furthermore, they show unbalanced chromosomal rearrangements (dicentrics, tricentrics, translocations) in the absence of any clastogenic agents ( Scappaticci et al, 1989 ; Dokal et al, 1992 ; Kehrer & Krone, 1992; Kehrer et al, 1992 ). In addition, peripheral blood and BM metaphases from some patients show numerous unbalanced chromosomal rearrangements in the absence of any clastogenic agents ( Dokal et al, 1992 ; Demiroglu et al, 1997 ). These studies provide evidence for a defect that predisposes DC cells to developing chromosomal rearrangements. DC, like FA, may thus be regarded as a chromosomal instability disorder, but with a predisposition to chromosomal rearrangements rather than the gaps and breaks seen in FA ( Dokal & Luzzatto, 1994). The demonstration of chromosomal instability suggests that cells of tissues with a high turnover, such as the BM, skin and gastrointestinal tract, may accumulate progressive DNA damage. This could in part account for the high frequency of BM failure and the epithelial abnormalities seen in these patients.

X-chromosome inactivation analysis in carriers of X-linked DC

X-chromosome inactivation patterns (XCIPs) have been studied in PB cells of women from X-linked DC families by investigating a methylation-sensitive restriction enzyme site in the polymorphic human androgen receptor locus at Xq11.2–Xq12 (HUMARA). All women known to be obligate carriers of DC showed complete skewing in XCIPs and, in addition, women predicted to be carriers on the basis of genetic analysis also had skewed XCIPs ( Devriendt et al, 1997 ; Ferraris et al, 1997 ; Vulliamy et al, 1997 ). The presence of the extremely skewed pattern of X inactivation in PB cells suggests that cells expressing the defective gene have a growth–survival disadvantage over those expressing the normal allele. Furthermore, a skewed XCIP provides important information about carrier status for use in the counselling of families at risk of DC. In addition, XCIP data may allow us to distinguish an inherited mutation from a de novo event in sporadic male DC cases, as well as autosomal from X-linked forms of the disease. This finding also suggests that X-linked carriers may have a reduced haemopoietic reserve and it will be important to see whether they are at a greater risk of developing BM failure than normal controls. Mild leucopenia has been observed in one X-linked carrier on the DCR ( Knight et al, 1998a )

von Willebrand factor levels

von Willebrand factor (VWF) antigen levels were raised in 7 out of 13 consecutive patients studied to date ( Dokal et al, 1995 ; unpublished observations). This suggests a predisposition to endothelial activation/damage. It is noteworthy that of the seven patients with raised VWF six had abnormalities of pulmonary diffusion capacity (reduced TLCO/KCO)/pulmonary disease; the six with normal VWF levels had normal diffusion capacity (unpublished observations) with three out of these six patients being below the age of 10 years. This preliminary observation may enable identification of patients who are more likely to develop pulmonary complications. VWF levels have been found to be significantly raised in patients who developed fatal vascular complications after SCT ( Berthou et al, 1991 ). The precise reason for this predisposition to endothelial damage is unclear. It could relate to a more general problem in all cells with high turnover or the endothelium may be the focus of some specific attack such as disregulated autoimmunity.

Positional cloning of the x-linked dc (dkc1) gene

The majority (86%) of patients with DC recruited on the DCR are male and provide further evidence that the X-linked recessive form of DC represents the majority of cases. Through linkage analysis in one large family, the DKC1 gene, responsible for X-linked DC, was mapped to Xq28 ( Connor & Teague, 1981; Connor et al, 1986 ). Three additional families confirmed this linkage, and a maximum logarithm of the odds ratio (LOD) score was obtained with DXS52 with no recombinations ( Arngrimsson et al, 1993 ). To refine further the localization of the DKC1 locus in Xq28, the families recruited in the DCR have been critical. Genetic linkage analysis in DCR multiplex families enabled the DKC1 candidate gene region to be defined more precisely. Initially, a 3·5-Mb DKC1 candidate region was defined between DXS1684 and DXS1108 in Xq28 ( Knight et al, 1996 ). Subsequently, the combined use of genetic linkage and XCIP analysis enabled this region to be narrowed to 1·4 Mb between Xq3274 and DXS1108 ( Knight et al, 1998b ). Hybridization screening with 28 positional candidate cDNAs from this region resulted in the detection of a 3' deletion in one DC patient and subsequent characterization of the gene responsible for X-linked dyskeratosis congenita DKC1 ( Heiss et al, 1998 ). The DKC1 gene is composed of 15 exons spanning 15 kb ( Hassock et al, 1999 ; Knight et al, 1999b ; Vulliamy et al, 1999a ); the DKC1 cDNA is 2·5 kb. The corresponding protein, dyskerin, is a protein of 514 amino acids with a predicted molecular weight of ∼ 57 kDa. It is highly conserved in eukaryotes. Significant homologies imply that DKC1 is the human orthologue of the yeast gene encoding centromere/microtubule binding protein Cbf5p, the rat gene encoding nucleolar protein NAP57 (Nopp140-associated protein) and Drosophila Nop60B/mfl gene ( Jiang et al, 1993 ; Meier & Blobel, 1994; Philips et al, 1998 ; Giordano et al, 1999 ). Functional studies of the rat NAP57 and yeast Cbf5p proteins suggest that dyskerin is a multifunctional protein involved in rRNA biosynthesis, ribosomal subunit assembly and/or centromere/microtubule binding. Furthermore, in dyskerin and its orthologues, a protein motif also present in the bacterial and yeast class of TruB pseudouridine (ψ) synthases was identified ( Koonin, 1996; Cadwell et al, 1997 ; Lafontaine et al, 1998 ). Pseudouridine synthases catalyse the isomerization of uridine to pseudouridine in non-coding RNAs. The Saccharomyces cerevisiae TruB homologues PUS4 and Cbf5p have been shown to possess pseudouridine synthase activity ( Becker et al, 1997 ; Zebarjadian et al, 1999 ) and thus dyskerin is predicted to be a member of the rRNA pseudouridine synthase group. Recent analysis using dyskerin tagged either with an immunoglobulin epitope ( Youssoufian et al, 1999 ) or with EGFP ( Heiss et al, 1999 ) or with a c-myc epitope (unpublished observations) have confirmed its localization to the nucleolus.

IMPACT OF RECENT ADVANCES and FUTURE DIRECTIONS

Identification of new variants of DC

The different abnormalities associated with DC are highly variable from patient to patient both in severity and age of onset. It is possible to think of each abnormality as a spectrum with there also being considerable heterogeneity in the combination of such abnormalities in a given patient. The severity of the DC phenotype may be defined by the age of onset of the BM failure and the number of associated somatic abnormalities; those with onset of BM failure below the age of 10 years in association with several somatic abnormalities can be regarded as having the severest phenotype, whereas those who develop haematological abnormalities after the age of 20 years have the mildest. It is emerging that those with early onset of BM failure die early and do not live long enough to develop some of the other complications of the disease. Thus, pulmonary complications, myelodysplasia and malignancy are seen in patients who live longer. It will be interesting to see whether it is possible to make genotype–phenotype correlations of prognostic significance. Furthermore, it is conceivable that DKC1 mutations may result in phenotypes which overlap with DC, but which hitherto have been classified into other disease categories. Indeed, one such example has already been identified.

The Hoyeraal–Hreidarsson (HH) syndrome is a severe multisystem disorder affecting boys characterized by microcephaly, cerebellar hypoplasia, growth retardation of prenatal onset, immunodeficiency and aplastic anaemia (AA) ( Hoyeraal et al, 1970 ; Hreidarsson et al, 1988 ; Berthet et al, 1994 ; Aalfs et al, 1995 ; Ohga et al, 1997 ; Nespoli et al, 1997 ). The observation that HH had been reported only in boys and the presence of AA in these patients raised the possibility that HH may be due to mutations in DKC1. To test whether HH was allelic to DC, the DKC1 gene was analysed in two HH families, one of which was the HH family described by Aalfs et al (1995). In one family, a nucleotide change at position 361 (A→G) in exon 5 (leading to an amino acid substitution of Ser→Gly at postion 121 in dyskerin) was found in both affected brothers; in the other family, a nucleotide change at position 146 (C→T) in exon 3 (leading to an amino acid substitution of Thr→Met at postion 47 in dyskerin) was found in the affected boys. The finding of these two novel missense mutations in two families with all the features of HH demonstrates that HH is a severe variant of X-linked DC ( Knight et al, 1999a ). It also shows that mutations in DKC1 can give rise to a much more heterogeneous clinical phenotype than previously thought, including cerebellar hypoplasia and severe immunodeficiency [particularly low/absent B lymphocytes and natural killer (NK) cells], which have not previously been regarded as an integral part of the DC phenotype. The severe developmental problems and cerebellar hypoplasia in HH families emphasize an important role of dyskerin in brain development.

The demonstration that HH is a severe variant of X-linked DC suggests that DKC1 mutations may contribute to a very wide clinical spectrum of patients. This may not have been recognized previously because clinical syndromes due to mutations in DKC1 that resulted in death (for example fatal infections because of severe immunodeficiency/AA) below the age of 10 years would not have been categorized as DC or HH as these patients did not live long enough to develop the diagnostic mucocutaneous features of classic DC or have all the features of HH. This suggests that children with severe clinical phenotypes who have some features of DC or HH (e.g. unexplained/idiopathic AA or immunodeficiency), but who lack the skin and nail changes, should now be screened for mutations in the DKC1 gene.

Based on the previous literature, the DCR data base and the recent demonstration that HH is a severe variant of X-linked DC, it is possible to draw the model shown in Fig 3. In this diagram, clinically recognizable dyskeratosis congenita has been grouped into X-linked and autosomal categories. Similarly, it is possible to identify X-linked and autosomal forms of HH. Indeed, a family with two female patients who had all the clinical features of HH have been recently reported ( Mahmood et al, 1998 ) and we are aware of other unpublished female cases of HH. Additionally, one can speculate that there are likely to be patients who do not clinically fit neatly into either classical DC or HH, but whose clinical features are due to mutations in DKC1 or in genes responsible for autosomal DC/HH. One such category is likely to be some ‘idiopathic aplastic anaemia’ patients. It is noteworthy that in five of the DCR families seven male members have died of severe AA (SAA) before the age of 8 years and the diagnosis of DC was made subsequently only when another member of the family survived long enough to develop the classic mucocutaneous features. One of these families (DCR009) has been published previously ( Forni et al, 1993 ) . There are also other reports in the literature in which patients were initially diagnosed to have bone marrow failure/AA and subsequently developed features of DC ( De Boeck et al, 1981 ; Phillips et al, 1992 ; Ivker et al, 1993 ).

Figure 3.

Model of clinical and genetic overlap between dyskeratosis congenita (DC) and Hoyeraal–Hreidarsson (HH) syndrome. The DC phenotype is shown in blue, split into autosomal and X-linked forms. The HH phenotype is shown in green which is also split into autosomal and X-linked forms. The disease caused by mutations in the DKC1 gene is shown by diagonal shading. The possibility for additional clinical phenotype(s) caused by mutations in DKC1 (but which does not clinically fit into either classic DC or HH) is shown in red.

Pathophysiology

The wide range of abnormalities seen in patients with HH and classic DC suggest that dyskerin has a functional role in many tissues and this is supported by the ubiquitous expression of the DKC1 gene ( Heiss et al, 1998 ). It is of interest that the worst affected tissues (skin and BM) have a high cell turnover. This clinical observation combined with the abnormalities of growth of fibroblasts, reduced haemopoietic progenitors and the skewed XCIPs in female carriers suggests that the DC gene may have a critical role in cell survival/proliferation; its deficiency having the greatest impact on cells with a high turnover. The precise function of DKC1 in the human cell and how mutations in this gene lead to the clinical phenotype, including the BM failure, is currently unknown. As highlighted above, homology searches imply that dyskerin is a multifunctional protein possibly involved in rRNA biosynthesis, ribosomal subunit assembly and/or centromere/microtubule binding. It is plausible that such a deficiency would have its maximal impact on tissues with a high turnover. Experiments will need to be designed to demonstrate this formally for dyskerin.

To date, we have identified DKC1 mutations in ∼ 40% of the DCR families. The majority of mutations ( Fig 4) identified have been missense ( Heiss et al, 1998 ; Knight et al, 1999b ; Rostamiani et al, 1999; Vulliamy et al, 1999a,b ). No null DKC1 mutations have been observed, suggesting that such mutations may be lethal. In yeast and Drosophila, null mutants have been shown to be lethal. It remains unclear how different mutations in the same gene can give rise to such varied clinical phenotypes. Further studies are needed to determine the functionally important residues of dyskerin and how different mutations affect its activity. It is noteworthy that the AGT→GGT mutation in exon 5 in one of the HH families, which can be regarded as the most severe phenotype clinically ( Knight et al, 1999a ), represents the first DKC1 mutation within the TruB domain ( Fig 5), which based on studies on the dyskerin yeast homologue Cbf5p is predicted to have a function in pseudouridine synthesis in pre-rRNA ( Heiss et al, 1998 ; Lafontaine et al, 1998 ; Zebarjadian et al, 1999 ). It is plausible that this mutation is associated with a greater defect in the pseudouridylation activity of dyskerin than mutations lying outside the TruB domains.

Figure 4.

Mutations in the DKC1 gene. This is a schematic representation of the genomic structure of the DKC1 gene with all the known mutations (bold) and polymorphisms (italics). The A353V mutation, marked with asterisk, is a recurrent de novo mutation which has been found in 17 different families. The mutations underlined (T49M, S121G) were detected in patients with the Hoyeraal–Hreidarsson syndrome. In addition to the missense mutations, two splicing mutations (IVS1 + 592 C > G; IVS2 −5C > G), a promoter mutation (URR −142C > G) and a 2-kb deletion are also marked.

Figure 5.

Schematic representation of dyskerin showing the locations of some possible function domains. The TruB (PSUS) domain is the catalytic domain of pseudouridine synthases which is shared with bacterial TruB proteins, yeast Pus4p and the Cb5fp family ( Gustafsson et al, 1996; Koonin, 1996). The PUA domian ( pseudo uridine synthases and archaeosine-specific transglycosylases) is a putative RNA binding domain ( Aravind & Koonin, 1999). NLS denotes the nuclear localization signals and poly-lysine the two lysine-rich carboxy domains.

It is also likely that dyskerin has other functions in addition to its role in pseudouridination. It is noteworthy that the dyskerin homologue Cbf5p has a centromere/microtubule binding domain and that some studies ( Scappaticci et al, 1989 ; Dokal et al, 1992 ; Kehrer & Krone, 1992; Kehrer et al, 1992 ; Dokal & Luzzatto, 1994; Demiroglu et al, 1997 ) have demonstrated chromosomal rearrangements (including dicentrics and tricentrics) in DC cells. This suggests that dyskerin may have a role in segregation of chromosomes. Additionally, it suggests a possible mechanism of how normal dyskerin may be important in coupling rRNA synthesis with cell division. Furthermore, abnormalities in segregation of chromosomes or synthesis of abnormal proteins and/or synthesis of normal proteins at reduced rates may be responsible for the increased predisposition to malignancy in DC.

Precursor rRNA transcripts undergo a number of post-transcriptional modifications before packaging with ribosomal proteins. This includes the pseudouridylation of selected uridine residues as mentioned above. This process is guided by a class of small nucleolar RNAs (snoRNAs) called the box H + ACA snoRNAs ( Ni et al, 1997 ). In yeast, the box H + ACA snoRNAs form a complex with a number of nucleolar proteins including Cbf5p, Gar1p, Nhp2p and Nop10p, of which Cbf5p is the core component ( Henras et al, 1998 ; Lafontaine et al, 1998 ; Watkins et al, 1998 ). The yeast Cbf5p and Drosophila Nop60B/mfl stabilize both the RNA and protein components of the box H + ACA complex and have been shown to be pseudouridine synthases. This functional role would therefore be predicted for dyskerin. It also suggests that some of the pathology in DC may arise from its inability to stabilize other H + ACA snoRNAs. In this regard, it is of interest that the RNA component (hTR) of telomerase contains the box H + ACA structural motif ( Mitchell et al, 1999a ). Dyskerin deficiency may thus lead to a problem in stabilizing telomerase, resulting in a secondary deficiency of telomerase activity. Indeed, Mitchell et al (1999b) have recently demonstrated that DC cell lines from two families have a lower level of telomerase RNA, produce lower levels of telomerase activity and have shorter telomeres than matched normal cells. We have studied fresh PB samples from 36 DC patients from 22 different families (unpublished observations) and have found that they also have significantly shorter telomere lengths than age-matched controls. A deficiency in telomerase could explain some of the clinical features of DC as compromised telomerase function leading to a defect in telomere maintenance may limit the proliferative capacity of somatic cells ( Colgin & Reddel, 1999) in the blood and epithelia. It also shows that different DKC1 mutations could produce very pleiotropic effects if different DKC1 mutations have varying effects on the stabilization of different H + ACA snoRNAs, of which telemorase is just one representative. Indeed, some features of the DC phenotype, such as the severe neurological abnormalities seen in the HH variant, are difficult to explain just on the basis of reduced telomerase activity. Further work is needed to substantiate the functional role of dyskerin and its interacting proteins and RNAs in the cell.

The skin pigmentation in DC patients can resemble very closely, clinically and histologically, the features of chronic graft-versus-host disease (GVHD) after allogeneic SCT ( Ling et al, 1985 ; Ivker et al, 1993 ). This suggests that although the primary defect in DC is a constitutional genetic abnormality (a putative inherited ribosomapathy; Luzzatto & Karadimitris, 1998), secondary pathology may in part be associated with immunological abnormalities that are observed in some DC patients ( Solder et al, 1998 ; Knight et al, 1999a ). Indeed, synthesis of abnormal proteins or abnormal rates of synthesis of normal proteins may lead to the presentation of antigens which, in turn, may incite autoimmune phenomena. Alternatively, the predicted defect in ribosomal biogenesis may explain the low B-lymphocyte and CD4 counts seen in some patients. The resulting disturbed CD4/CD8 ratio may lead to altered immune responses, including those against host antigens.

Families with affected females

Three female carriers of X-linked DC had mild clinical features (single dystrophic nail, a discrete area of pigmentation, mild leucopenia and lumbar scoliosis) and the clinical phenotype in some female patients is less severe ( Knight et al, 1998a ). This raises the possibility that some of the female patients who have hitherto been classified into the autosomal forms of DC may turn out to have mutations in DKC1. However, to date, we have not found DKC1 mutations in any female cases (unpublished observations). This substantiates the case for autosomal DC loci.

Towards haemopoietic gene therapy

Treatment for DC patients developing SAA remains unsatisfactory ( Dokal, 1996b) as, even for the small subset of patients who have compatible sibling donors, the results are very poor because of a high incidence of fatal pulmonary complications. There is therefore a clinical need to develop new treatment strategies for patients developing SAA. As DC is a single gene recessive disorder and the cells that need to be targeted (haemopoietic stem cells) are accessible, DC is a good candidate for haemopoietic gene therapy. Furthermore, there is evidence from fibroblast culture studies and from skewed X-chromosome inactivation patterns (XCIPs) in DC carriers that cells transfected with the normal gene would have growth/survival advantage compared with the uncorrected cells. Such an advantage would also be predicted from the putative role of dyskerin in ribosome biogenesis.

Haemopoietic studies in DC suggest there is ‘premature ageing’ of the haemopoietic system; there is a reduction in the proliferative potential of clonogenic haemopoietic progenitors and a reduced subplating cloning efficiency. Further characterization of DKC1 will facilitate construction of vectors capable of expressing wild-type DKC1 in haemopoietic cells. Successful correction of DC haemopoietic cells in vitro will form the basis of developing a protocol of haemopoietic gene therapy for DC patients. The precise strategies used for these final studies will depend on technical advances in vectors and transfection that are being intensively pursued in many laboratories. In addition, information on the regulation of the DKC1 and the availability of a DC mouse model may facilitate the development of an efficient haemopoietic gene therapy strategy. These experiments may also have important implications for the management of patients with idiopathic AA.

Relationship to idiopathic aplastic anaemia

Dyskerin represents the first protein to be identified that is important in the primary pathology of AA and where homology searches have suggested a possible function for the protein. To what extent might ribosome biogenesis be disrupted in idiopathic AA patients? Nucleolar events in which dyskerin is involved are currently under intense investigation. Proteins which interact with dyskerin may turn out to be important targets in the biology not only of autosomal DC/HH but also of some types of idiopathic AA.

Conclusion

DC is a severe multisystem disorder associated with premature mortality usually due to BM failure/immunodeficiency for which present forms of treatment are unsatisfactory. The majority of patients are men showing a X-linked recessive pattern of inheritance. The identification of the DKC1 gene and its corresponding nucleolar protein, dyskerin, provides an accurate diagnostic test that now facilitates early diagnosis, especially in patients presenting with atypical features such as the Hoyeraal–Hreidarsson syndrome; patients with unexplained aplastic anaemia or immunodeficiency and who are below the age of 10 years should be screened for DC. The task over the next few years is to determine the function of dyskerin in the normal and diseased cell. The DKC1 gene also provides a ‘handle’ to identify the proteins (and their corresponding genes) responsible for autosomal DC/HH and some subtypes of ‘idiopathic’ aplastic anaemia or immunodeficiency. As homology searches have suggested that dyskerin has a putative function in ribosome biogenesis, this advance may shape our understanding of not only the AA associated with DC but also that arising in other patients with hitherto called ‘idiopathic’ AA. This may, in turn, lead to new treatment strategies for AA patients who cannot be treated by haemopoietic stem cell transplantation.

Acknowledgments

I am indebted to Stuart Knight, Philip Mason and Tom Vulliamy, whose contributions has been critical over the past 5 years. I thank my other current (Andy Chase, John Goldman, Myrtle Gordon, Samia Hawisa, Jaspal Kaeda, Mike Laffan, Richard Manning, Steve Marley, Irene Roberts, David Stevens) and past (Lucio Luzzatto) colleagues at the Hammersmith Hospital, who have contibuted and continue to contribute towards the clinical and scientific aspects of the DC project. I am also grateful to all my colleagues for their support in establishing the dyskeratosis congenita registry (DCR) and to the Wellcome Trust and Action Research for their financial support.

Ancillary