Download - Dyskeratosis congenita in all its forms

768 q 2000 Blackwell Science Ltd

Review

DYSKERATOSIS CONGENITA IN ALL ITS FORMS

Classic dyskeratosis congenita (DC) is an inherited diseasecharacterized 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, neuro-logical, ophthalmic, pulmonary and skeletal) abnormalitieshave also been reported (Sirinavin & Trowbridge, 1975;Drachtman & Alter, 1995; Dokal, 1996a; Knight et al,1998a). Bone marrow (BM) failure is the principal cause ofearly mortality with an additional predisposition to malig-nancy and fatal pulmonary complications. X-linked recessive,autosomal dominant and autosomal recessive forms of thedisease are recognized.

Since the annotation (Dokal, 1996a), there have beensignificant advances in DC. These have been facilitated bythe dyskeratosis congenita registry (DCR), established at theHammersmith Hospital (London) in 1995. By November1999, 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 familiesfrom 20 different countries collectively comprised 148 (127male and 21 female) patients. As well as confirming previ-ous observations, the DCR has identified new features of DCand has been pivotal in the identification of the DKC1 genewhich is mutated in X-linked DC.

CLINICAL ASPECTS

In 76 out of the 92 families, there were only males affectedand these collectively comprised 118 patients. In 25 of these76 families, there were two or more affected males with lackof male-to-male transmission, consistent with an X-linkedrecessive pattern of inheritance. In the 51 families withaffected sporadic males, it is likely that many of these alsorepresent the X-linked form of the disease, although somemay represent autosomal forms of DC. Overall, out of the148 patients, 127 (86%) were male and this confirmsprevious reports that the major form of DC is X linked.

In 16 families out of the total of 92, there was one ormore affected female. In 4 of these 16, the cases weresporadic with a history of parental consanguinity in two; ineight families, there were two affected members in the samegeneration with a history of parental consanguinity in two;

in three families, there were affected cases in two differentgenerations; finally, in one out of the 16 families, the twocases were first maternal cousins. Thus, collectively they arelikely to represent different genetic subtypes. Some of theseare likely to represent autosomal recessive forms of thedisease and others autosomal dominant. These familiestherefore provide further evidence for autosomal recessiveand dominant forms of the disease in addition to thosepublished 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 onlySomatic abnormalities. A wide range of somatic abnorm-

alities were seen as listed in Table I. DC may therefore beregarded as an inherited multisystem syndrome. In general,the abnormalities were not neonatal in manifestation, butdeveloped progressively at a variable rate. The muco-cutaneous features (skin pigmentation, nail dystrophy andleucoplakia; Fig 1) usually appeared between the ages of 5and 10 years. The median ages of onset for abnormal skinpigmentation, nail dystrophy and leucoplakia were 8 years(range 0´5±21 years), 6 years (range 1±17 years) and7 years (range 1±26 years) respectively. There was a wideage range over which these features developed and therewere also significant qualitative differences in the skinpigmentation and the nail dystrophy; for example, somepatients had a very florid rash involving most of the skin,others only had a localized rash and some patients hadminimal nail changes, whereas some developed completenail loss. In families with two or more affected members, thephenotypes among the different individuals tended to besimilar. However, in some instances there was significantvariability in the severity of the clinical phenotype indifferent members of the same family. This suggests that thephenotype may be modified by other genetic and/orenvironmental factors.

A subset (20´3%) of patients developed pulmonarycomplications (Table I) with reduced diffusion capacityand/or restrictive defect. It has been possible to studysome of these patients at several time points. For example, inone patient (aged 36 years) the diffusion capacity (TLCO)fell from 73% to 59% over a period of 6 months, suggestingthat pulmonary abnormalities also progress with age andhighlighting the need for regular monitoring. Postmortemstudies on two patients who died suddenly from acuterespiratory disease showed abnormal levels of pulmonary

British Journal of Haematology, 2000, 110, 768±779

Correspondence: Dr Inderjeet Dokal, Department of Haematology,Imperial College School of Medicine (ICSM), Hammersmith Campus,

Du Cane Road, London W2 0NN, UK. E-mail: [email protected]

fibrosis and abnormalities in the pulmonary microvascu-lature. These histological changes correlate with theabnormalities in fibroblasts (Scappaticci et al, 1989; Dokalet al, 1992; Kehrer & Krone, 1992; Kehrer et al, 1992)observed in DC skin biopsies and the telangiectatic vesselsseen at the skin surface clinically. The development ofpulmonary abnormalities highlighted by the DCR andprevious reports (Paul et al, 1992; Verra et al, 1992) may

in part explain the high incidence of early and late fatalpulmonary complications after bone marrow transplanta-tion (BMT) (Berthou et al, 1991; Dokal et al, 1992; Langstonet al, 1996; Yabe et al, 1997; Rocha et al, 1998).

Haematological abnormalities. Bone marrow failure result-ing in peripheral cytopenias appears to be much morefrequent than previously thought. As can be seen fromTable II, 85´5% of patients had a peripheral cytopenia ofone or more lineages, with 76´3% having a cytopenia of twoor more lineages; in 80% of the patients who developedpancytopenia, the age of onset was less than 20 years(median 8 years), with 50% developing pancytopenia belowthe age of 10 years. Accepting that there may be some biasin the patients referred to the DCR, the actual probability ofdeveloping BM failure [one or more peripheral cytopenia(s)]is much higher than previously documented, approaching94% by the age of 40 years (Fig 2). This is also reflected inthe causes of death (see below). It is noteworthy that onepatient (aged 29 years) had approximately 10% myeloidblasts in the BM (Dokal et al, 1992) and three others hadhypocellular 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 BMthere is a predisposition to both MDS and acute myeloidleukaemia (AML) in patients with DC.

Families with affected femalesThe 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 andnine male cases. Several of the men had features that weresimilar to those seen in families with affected males only (seeabove). Indeed, in two families (DCR019 and DCR024), thediagnosis of DC in the female members was facilitated by thediagnosis of the affected male patient.

Table I. Somatic abnormalities/complications in DC families with

affected males only (n � 118).

Abnormality % of patients

Abnormal skin pigmentation 89´0

Nail dystrophy 88´0

Bone marrow failure 85´5

Leucoplakia 78´0Epiphora 30´5

Learning difficulties/developmental delay/

±mental retardation

25´4

Pulmonary disease 20´3

Short stature 19´5

Extensive dental caries/loss 16´9

Oesophageal stricture 16´9Hair loss/grey hair/sparse eyelashes 16´1

Hyperhiderosis 15´3

Malignancy 7´6

Intrauterine growth retardation 7´6Liver disease/peptic ulceration/enteropathy 7´3

Ataxia 6´8

Hypogonadism/undescended testes 5´9Microcephaly 5´9

Urethral stricture/phimosis 5´1

Osteoporosis/aseptic necrosis/scoliosis 5´1

Deafness 0´8

Fig 1. Photographs of DC patients showing abnormal skin pigmentation (A, B, C and D), nail dystrophy of finger nails (E) and toe nails (F andG) and leucoplakia of tongue (H).

q 2000 Blackwell Science Ltd, British Journal of Haematology 110: 768±779

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The phenotype in the female cases varied considerably. Infamily DCR014, the diagnosis in the case of the sporadicfemale case was based on the presence of the muco-cutaneous triad associated with aplasia below the age of10 years. In family DCR019, the female case was thedaughter of a male patient. She developed BM failure at theage of 4 years without cutaneous features. In familyDCR022, both females developed BM failure, they hadfewer than two nails affected and had no skin pigmentation.In family DCR024, the female case died of BM failure aged8 years; her brother was alive and had all the classicfeatures seen in the male patients (see above). In familyDCR028, the patient had skin pigmentation abnormalities,leucoplakia, thrombocytopenia and carcinoma of thelarynx, but she had no nail dystrophy at the age of46 years. In family DCR039 (previously published by Elliottet al, 1999), the male patient died from BM failure aged8 years; his sister had mild skin pigmentation, leucoplakiaand nail dystrophy. The parents of these two siblings werefirst cousins. In family DCR063, the 12-year-old girl hadreticulate skin pigmentation, leukoplakia and hypocellularBM but no nail dystrophy; her 10-year-old brother had naildystrophy as well as all the other features present in hisolder sister. In family DCR070, there were two affectedsisters and one affected brother in one generation and theirparents were first cousins. In family DCR073, the affected3-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 inwhich there were two affected sisters with early onset of BMfailure (associated with leucoplakia/tongue ulceration,short stature and ataxia), one of whom died from sepsisaged 2 years. In family DCR082, the affected members werea mother and daughter; the daughter had skin pigmenta-tion and leucoplakia but no nail dystrophy, whereas themother (aged 52 years) had skin pigmentation, macrocyticanaemia and a low CD4 count that had been associatedwith Pneumocystis carinii pneumonia. These two womentherefore had mild disease which appeared to be segregatingas an autosomal dominant trait. In family DCR083, therewere three affected members in two generations, a sister andbrother and their father. The sister died aged 30 years fromrespiratory complications; she had skin pigmentation,pancytopenia and squamous carcinoma of the tongue. Herbrother, aged 47 years, had aplastic anaemia, severe acneand absent fingerprints. Their father was transfusiondependent and is said to have died from iron overload.The three members in this family had mild mucocutaneousfeatures and appeared to show autosomal dominanttransmission of the clinical phenotype. In family DCR086,there was one affected girl aged 2 years and her parentswere first cousins. She had severe intrauterine growth retarda-tion, microcephaly, developmental delay, ataxia, leucoplakiaand severe BM failure at age 2 years. In DCR087, there werethree affected members in one sibship, two sisters and theirbrother. They all had peripheral cytopenia, the brother alsohad significant atrioventricular (AV) malformation. In familyDCR088, the two affected members were first cousins. Theindex case was a boy who presented at age 4 years with BMfailure. He subsequently developed reticulate skin pigmenta-tion, thin hair and extensive dental caries. At the age of18 years, he still had no nail dystrophy and no leucoplakia.His maternal cousin had aplastic anaemia which respondedto steroids and she had very thin hair.

It can be seen that the clinical features in these familieswith female cases are very variable. In general, in families inwhich the inheritance appears to be autosomal recessive(e.g. DCR022, DCR024, DCR039, DCR 070, DCR079 andDCR086), the phenotype is more severe than in those inwhich it appears to be autosomal dominant (e.g. DCR019,DCR082 and DCR083). This agrees broadly with previousliterature (autosomal recessive: Sorrow & Hitch, 1963; Linget 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 recessiveand dominant forms of DC.

Causes of deathAnalysis from the DCR shows that 67% of deaths resulteddirectly from BM failure or from complications of itstreatment. Nine per cent died from sudden pulmonarycomplications. In a further 9%, fatal pulmonary complica-tions were seen in the context of a BMT. Six per cent diedfrom malignancy and 9% died of causes unrelated to DC andits treatment (rabies, myocardial infarction, road trafficaccident). The majority of deaths were due to infection and

100

80

60

40

20

0

Pro

bab

ility

(%

)

0Patient age in years

10 20 30 40

94%

Fig 2. Probability of bone marrow failure in dyskeratosis con-

genita.

Table II. Haematological complications in DC families with

affected males only.

Haematological abnormality % of patients

Pancytopenia 76´3

Thrombocytopenia only 6´6

Leucopenia only 2´6

No cytopenia 14´5

Median age for onset of pancytopenia was 10 years.

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these usually occurred (, 80%) before the end of thesecond decade of life. It is becoming increasingly recog-nized from patients on the DCR and from the previousreports that immunological abnormalities (includingreduced or elevated immunoglobulin levels, reduced B-and/or T-lymphocyte numbers and reduced or absentresponses to phytohaemagglutinin) can occur in a sub-group of patients with or without associated BM hypo-plasia. Some deaths from infection may thus be attributableto immunodeficiency rather than aplastic anaemia. Fatalopportunistic infections such as Pneumocystis cariniipneumonia and cytomegalovirus have been previouslyreported (Wiedemann et al, 1984; Rose & Kern, 1992) andthis aspect, with special consideration of immunologicalabnormalities (see Table III), was recently reviewed by Solderet al (1998).

MalignancyMalignancies developed in 13 (8´8%) out of the 148patients. These included four cases of myelodysplasia (twopatients with refractory anaemia and two patients withrefractory anaemia with excess blasts), one which developedat age 10, one at 22, one at 27 and one at 29 years. Therewas also one case of Hodgkin's lymphoma (25 years) andeight cases of carcinoma: bronchus (56 years), colon(20 years), larynx (47 years), oesophagus (38 years), pan-creas (29 years), skin (20 years) and tongue (one at 30 andone at 38 years). Of the 13 malignancies, three were infemale patients (one case of MDS and two cases ofcarcinoma) and the majority of malignancies developed inthe third decade or later.

Current treatments for the bone marrow failureTreatment for severe BM failure in DC remains unsatis-factory. As in Fanconi's anaemia, the anabolic steroidoxymetholone can produce an improvement in haemo-poietic function in many patients for a variable period oftime (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 alsobeen reported (Russo et al, 1990; Alter et al, 1997). Themain treatment for severe aplastic anaemia (SAA), however,is allogeneic stem cell transplantation (SCT) and there issome experience of using both sibling and alternative stemcell donors. To date, SCT has been performed in eight of thepatients on the registry and some of these details have beenpublished 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 whomhad sibling donors (one now at 4, one at 5 and one at7 years after SCT) and the fourth had an unrelated SCT3 years ago. There are also other reports of the use of SCT inpatients with DC with some long-term survivors (Philipset al, 1992; Langston et al, 1996; Yabe et al, 1997;Ghavamzadeh et al, 1999). Unfortunately, because of earlyand 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 lesssuccessful than in FA. The presence of pulmonary diseasein a significant proportion of DC patients (Table I) perhapsnow explains the high incidence of fatal pulmonarycomplications in the setting of SCT. It also highlights theneed to avoid drugs which are associated with pulmonarytoxicity (such as busulphan) and to use pulmonaryshielding if radiotherapy is used for SCT. As BM failure isthe main cause of premature death in DC patients and SCTis currently the only curative option for the BM failure (withsome long-term survivors), SCT should continue to beperformed on carefully selected patients. Perhaps the bestcandidates for SCT are patients with no pre-existingpulmonary disease and who have sibling donors. SCTusing fludarabine-based protocols appears to be givingencouraging results in FA patients and may be worthexploring in patients with DC. There is a great need todevelop new and better treatment strategies for DC patientswith SAA.

Table III. Immunological data on 67 patients with DC.

Immunoglobulin levels Normal (%) Elevated (%) Reduced (%)

IgG (n = 53) 24/53 (45) 20/53 (38) 9/53 (17)

IgA (n = 54) 29/54 (54) 21/54 (39) 4/54 (7)

IgM (n = 53) 34/53 (64) 10/53 (19) 9/53 (17)Lymphocyte counts Normal (%) Elevated (%) Reduced (%)

B lymphocytes (n = 17) 7/17 (41) 0 10/17 (59)

T lymphocytes (n = 21)Total T±lymphocytes (n = 17) 7/17 (41) 1/17 (6) 9/17 (53)

CD4 cell count (n = 6) 2/6 (33) 0 4/6 (67)

CD4/CD8 ratio (n = 5) 2/5 (40) 0 3/5 (60)

Stimulation of lymphocytes by PHA(n = 17) Normal (%) Absent/reduced (%)7/17 (41) 10/17 (59)

Sensitivity to skin antigens(n = 18) Normal (%) Absent/reduced (%)

3/18 (17) 15/18 (83)

This includes the data reviewed by Solder et al (1998) and a further 21 patients.

PHA, phytohaemagglutinin.


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DC CELL PHENOTYPE

Haemopoietic progenitorsHaemopoietic progenitor studies (Colvin et al, 1984; Fried-land et al, 1985; Alter et al, 1992; Dokal et al, 1992; Marshet al, 1992) have shown reduced numbers of all progenitors(erythroid, myeloid and megakaryocytic) compared withcontrols and there is usually a downward decline with time.The degree to which the progenitors are reduced can varyfrom patient to patient and they can be reduced even whenthe PB count is normal (unpublished observations). Thehaemopoietic system appears to undergo `premature ageing'with a reduction in the proliferative potential of clonogenicprogenitors and a reduced subplating cloning efficiency(Marley et al, 1999; unpublished data). The demonstrationof abnormalities of growth and chromosomal rearrange-ments in fibroblasts suggests that the BM failure is likely tobe a consequence of abnormalities in both haemopoieticstem cells (Friedland et al, 1985; Marsh et al, 1992) andstromal cells.

Chromosomal instabilityDC has many features in common with Fanconi's anaemiain which there is a hypersensitivity to clastogenic agentssuch as mitomycin C (MMC), which is a DNA alkylator.Some authors have reported excessive chromosome break-age 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 & Trow-bridge, 1975; Drachtman & Alter, 1992) with the resultthat, until recently, confusion existed over the susceptibilityof DC cells to clastogenic agents. Coulthard et al (1998)chose a range of agents, based on previous reportssuggesting susceptibility, to determine which agents wouldproduce an increased level of breakage in DC lymphocytesover that seen in normal controls. This study demonstratedthat there was no significant difference in chromosomalbreakage between DC and normal lymphocytes with orwithout the use of bleomycin, diepoxybutane (DEB), MMCand g-irradiation. This conclusive study now enables DCpatients to be distinguished from FA.

Primary DC skin fibroblasts are abnormal both inmorphology and in growth rate. Furthermore, they showunbalanced chromosomal rearrangements (dicentrics, tri-centrics, translocations) in the absence of any clastogenicagents (Scappaticci et al, 1989; Dokal et al, 1992; Kehrer &Krone, 1992; Kehrer et al, 1992). In addition, peripheralblood and BM metaphases from some patients shownumerous unbalanced chromosomal rearrangements inthe absence of any clastogenic agents (Dokal et al, 1992;Demiroglu et al, 1997). These studies provide evidence for adefect that predisposes DC cells to developing chromosomalrearrangements. DC, like FA, may thus be regarded as achromosomal instability disorder, but with a predispositionto chromosomal rearrangements rather than the gaps andbreaks seen in FA (Dokal & Luzzatto, 1994). The demon-stration of chromosomal instability suggests that cells oftissues with a high turnover, such as the BM, skin andgastrointestinal tract, may accumulate progressive DNA

damage. This could in part account for the high frequencyof BM failure and the epithelial abnormalities seen in thesepatients.

X-chromosome inactivation analysis in carriers of X-linked DCX-chromosome inactivation patterns (XCIPs) have beenstudied in PB cells of women from X-linked DC families byinvestigating a methylation-sensitive restriction enzyme sitein the polymorphic human androgen receptor locus atXq11.2±Xq12 (HUMARA). All women known to be obligatecarriers of DC showed complete skewing in XCIPs and, inaddition, women predicted to be carriers on the basis ofgenetic analysis also had skewed XCIPs (Devriendt et al,1997; Ferraris et al, 1997; Vulliamy et al, 1997). Thepresence of the extremely skewed pattern of X inactivationin PB cells suggests that cells expressing the defective genehave a growth±survival disadvantage over those expressingthe normal allele. Furthermore, a skewed XCIP providesimportant information about carrier status for use in thecounselling of families at risk of DC. In addition, XCIP datamay allow us to distinguish an inherited mutation from a denovo event in sporadic male DC cases, as well as autosomalfrom X-linked forms of the disease. This finding also suggeststhat X-linked carriers may have a reduced haemopoieticreserve and it will be important to see whether they are at agreater risk of developing BM failure than normal controls.Mild leucopenia has been observed in one X-linked carrieron the DCR (Knight et al, 1998a)

von Willebrand factor levelsvon Willebrand factor (VWF) antigen levels were raised in 7out of 13 consecutive patients studied to date (Dokal et al,1995; unpublished observations). This suggests a predis-position to endothelial activation/damage. It is noteworthythat of the seven patients with raised VWF six hadabnormalities of pulmonary diffusion capacity (reducedTLCO/KCO)/pulmonary disease; the six with normal VWFlevels had normal diffusion capacity (unpublished observa-tions) with three out of these six patients being below theage of 10 years. This preliminary observation may enableidentification of patients who are more likely to developpulmonary complications. VWF levels have been found to besignificantly raised in patients who developed fatal vascularcomplications after SCT (Berthou et al, 1991). The precisereason for this predisposition to endothelial damage isunclear. It could relate to a more general problem in all cellswith high turnover or the endothelium may be the focus ofsome specific attack such as disregulated autoimmunity.

POSITIONAL CLONING OF THE X-LINKED DC(DKC1) GENE

The majority (86%) of patients with DC recruited on theDCR are male and provide further evidence that the X-linkedrecessive form of DC represents the majority of cases.Through linkage analysis in one large family, the DKC1gene, responsible for X-linked DC, was mapped to Xq28(Connor & Teague, 1981; Connor et al, 1986). Threeadditional families confirmed this linkage, and a maximum

772 Review


logarithm of the odds ratio (LOD) score was obtained withDXS52 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. Geneticlinkage analysis in DCR multiplex families enabled the DKC1candidate gene region to be defined more precisely. Initially,a 3´5-Mb DKC1 candidate region was defined betweenDXS1684 and DXS1108 in Xq28 (Knight et al, 1996).Subsequently, the combined use of genetic linkage and XCIPanalysis enabled this region to be narrowed to 1´4 Mbbetween Xq3274 and DXS1108 (Knight et al, 1998b).Hybridization screening with 28 positional candidatecDNAs from this region resulted in the detection of a 3'deletion in one DC patient and subsequent characterizationof the gene responsible for X-linked dyskeratosis congenitaDKC1 (Heiss et al, 1998). The DKC1 gene is composed of 15exons 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 514amino acids with a predicted molecular weight of , 57 kDa.It is highly conserved in eukaryotes. Significant homologiesimply that DKC1 is the human orthologue of the yeast geneencoding centromere/microtubule binding protein Cbf5p,the rat gene encoding nucleolar protein NAP57 (Nopp140-associated protein) and Drosophila Nop60B/mfl gene (Jianget al, 1993; Meier & Blobel, 1994; Philips et al, 1998;Giordano et al, 1999). Functional studies of the rat NAP57and yeast Cbf5p proteins suggest that dyskerin is amultifunctional protein involved in rRNA biosynthesis,ribosomal subunit assembly and/or centromere/micro-tubule binding. Furthermore, in dyskerin and its ortho-logues, a protein motif also present in the bacterial andyeast class of TruB pseudouridine (c) synthases wasidentified (Koonin, 1996; Cadwell et al, 1997; Lafontaineet al, 1998). Pseudouridine synthases catalyse the isomer-ization of uridine to pseudouridine in non-coding RNAs. TheSaccharomyces cerevisiae TruB homologues PUS4 and Cbf5phave been shown to possess pseudouridine synthase activity

(Becker et al, 1997; Zebarjadian et al, 1999) and thusdyskerin is predicted to be a member of the rRNA pseudo-uridine synthase group. Recent analysis using dyskerintagged either with an immunoglobulin epitope (Youssoufianet al, 1999) or with EGFP (Heiss et al, 1999) or with a c-mycepitope (unpublished observations) have confirmed itslocalization to the nucleolus.

IMPACT OF RECENT ADVANCES AND FUTUREDIRECTIONS

Identification of new variants of DCThe different abnormalities associated with DC are highlyvariable from patient to patient both in severity and age ofonset. It is possible to think of each abnormality as aspectrum with there also being considerable heterogeneityin the combination of such abnormalities in a given patient.The severity of the DC phenotype may be defined by the ageof onset of the BM failure and the number of associatedsomatic abnormalities; those with onset of BM failure belowthe age of 10 years in association with several somaticabnormalities can be regarded as having the severestphenotype, whereas those who develop haematologicalabnormalities after the age of 20 years have the mildest. Itis emerging that those with early onset of BM failure dieearly and do not live long enough to develop some of theother complications of the disease. Thus, pulmonarycomplications, myelodysplasia and malignancy are seen inpatients who live longer. It will be interesting to see whetherit is possible to make genotype±phenotype correlations ofprognostic significance. Furthermore, it is conceivable thatDKC1 mutations may result in phenotypes which overlapwith DC, but which hitherto have been classified into otherdisease categories. Indeed, one such example has alreadybeen identified.

The Hoyeraal±Hreidarsson (HH) syndrome is a severemultisystem disorder affecting boys characterized by micro-cephaly, cerebellar hypoplasia, growth retardation of

Fig 3. Model of clinical and genetic overlap

between dyskeratosis congenita (DC) and

Hoyeraal±Hreidarsson (HH) syndrome. The

DC phenotype is shown in blue, split intoautosomal 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 phe-

notype(s) caused by mutations in DKC1 (butwhich does not clinically fit into either

classic DC or HH) is shown in red.


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prenatal onset, immunodeficiency and aplastic anaemia(AA) (Hoyeraal et al, 1970; Hreidarsson et al, 1988; Berthetet al, 1994; Aalfs et al, 1995; Ohga et al, 1997; Nespoli et al,1997). The observation that HH had been reported only inboys and the presence of AA in these patients raised thepossibility that HH may be due to mutations in DKC1. Totest whether HH was allelic to DC, the DKC1 gene wasanalysed in two HH families, one of which was the HHfamily described by Aalfs et al (1995). In one family, anucleotide change at position 361 (A!G) in exon 5 (leadingto an amino acid substitution of Ser!Gly at postion 121 indyskerin) was found in both affected brothers; in the otherfamily, a nucleotide change at position 146 (C!T) in exon 3(leading to an amino acid substitution of Thr!Met atpostion 47 in dyskerin) was found in the affected boys. Thefinding of these two novel missense mutations in twofamilies with all the features of HH demonstrates that HH isa severe variant of X-linked DC (Knight et al, 1999a). It alsoshows that mutations in DKC1 can give rise to a much moreheterogeneous clinical phenotype than previously thought,including cerebellar hypoplasia and severe immunodefi-ciency [particularly low/absent B lymphocytes and naturalkiller (NK) cells], which have not previously been regardedas an integral part of the DC phenotype. The severedevelopmental problems and cerebellar hypoplasia in HHfamilies emphasize an important role of dyskerin in braindevelopment.

The demonstration that HH is a severe variant of X-linkedDC suggests that DKC1 mutations may contribute to a verywide clinical spectrum of patients. This may not have beenrecognized previously because clinical syndromes due tomutations in DKC1 that resulted in death (for example fatalinfections because of severe immunodeficiency/AA) belowthe age of 10 years would not have been categorized as DCor HH as these patients did not live long enough to developthe diagnostic mucocutaneous features of classic DC or haveall the features of HH. This suggests that children withsevere clinical phenotypes who have some features of DC orHH (e.g. unexplained/idiopathic AA or immunodeficiency),but who lack the skin and nail changes, should now bescreened for mutations in the DKC1 gene.

Based on the previous literature, the DCR data base andthe recent demonstration that HH is a severe variant of X-linked DC, it is possible to draw the model shown in Fig 3. Inthis diagram, clinically recognizable dyskeratosis congenitahas been grouped into X-linked and autosomal categories.Similarly, it is possible to identify X-linked and autosomalforms of HH. Indeed, a family with two female patients whohad all the clinical features of HH have been recentlyreported (Mahmood et al, 1998) and we are aware of otherunpublished female cases of HH. Additionally, one canspeculate that there are likely to be patients who do notclinically fit neatly into either classical DC or HH, but whoseclinical features are due to mutations in DKC1 or in genesresponsible for autosomal DC/HH. One such category islikely to be some `idiopathic aplastic anaemia' patients. It isnoteworthy that in five of the DCR families seven malemembers have died of severe AA (SAA) before the age of8 years and the diagnosis of DC was made subsequently

only when another member of the family survived longenough to develop the classic mucocutaneous features. Oneof these families (DCR009) has been published previously(Forni et al, 1993). There are also other reports in theliterature in which patients were initially diagnosed to havebone marrow failure/AA and subsequently developedfeatures of DC (De Boeck et al, 1981; Phillips et al, 1992;Ivker et al, 1993).

PathophysiologyThe wide range of abnormalities seen in patients with HHand classic DC suggest that dyskerin has a functional role inmany tissues and this is supported by the ubiquitousexpression of the DKC1 gene (Heiss et al, 1998). It is ofinterest that the worst affected tissues (skin and BM) have ahigh cell turnover. This clinical observation combined withthe abnormalities of growth of fibroblasts, reduced haemo-poietic progenitors and the skewed XCIPs in female carrierssuggests that the DC gene may have a critical role in cellsurvival/proliferation; its deficiency having the greatestimpact on cells with a high turnover. The precise function ofDKC1 in the human cell and how mutations in this genelead to the clinical phenotype, including the BM failure, iscurrently unknown. As highlighted above, homologysearches imply that dyskerin is a multifunctional proteinpossibly involved in rRNA biosynthesis, ribosomal subunitassembly and/or centromere/microtubule binding. It isplausible that such a deficiency would have its maximalimpact on tissues with a high turnover. Experiments willneed to be designed to demonstrate this formally fordyskerin.

To date, we have identified DKC1 mutations in , 40% ofthe DCR families. The majority of mutations (Fig 4) identi-fied have been missense (Heiss et al, 1998; Knight et al,1999b; Rostamiani et al, 1999; Vulliamy et al, 1999a,b). Nonull DKC1 mutations have been observed, suggesting thatsuch mutations may be lethal. In yeast and Drosophila, nullmutants have been shown to be lethal. It remains unclearhow different mutations in the same gene can give rise tosuch varied clinical phenotypes. Further studies are neededto determine the functionally important residues of dyskerinand how different mutations affect its activity. It is note-worthy that the AGT!GGT mutation in exon 5 in one of theHH families, which can be regarded as the most severephenotype clinically (Knight et al, 1999a), represents thefirst DKC1 mutation within the TruB domain (Fig 5), whichbased on studies on the dyskerin yeast homologue Cbf5p ispredicted to have a function in pseudouridine synthesis inpre-rRNA (Heiss et al, 1998; Lafontaine et al, 1998;Zebarjadian et al, 1999). It is plausible that this mutationis associated with a greater defect in the pseudouridylationactivity of dyskerin than mutations lying outside the TruBdomains.

It is also likely that dyskerin has other functions inaddition to its role in pseudouridination. It is noteworthythat the dyskerin homologue Cbf5p has a centromere/microtubule binding domain and that some studies (Scap-paticci et al, 1989; Dokal et al, 1992; Kehrer & Krone, 1992;Kehrer et al, 1992; Dokal & Luzzatto, 1994; Demiroglu et al,

774 Review


1997) have demonstrated chromosomal rearrangements(including dicentrics and tricentrics) in DC cells. Thissuggests that dyskerin may have a role in segregation ofchromosomes. Additionally, it suggests a possible mechan-ism of how normal dyskerin may be important in couplingrRNA synthesis with cell division. Furthermore, abnor-malities in segregation of chromosomes or synthesis ofabnormal proteins and/or synthesis of normal proteins atreduced rates may be responsible for the increased predis-position to malignancy in DC.

Precursor rRNA transcripts undergo a number of post-transcriptional modifications before packaging with ribo-somal proteins. This includes the pseudouridylation ofselected uridine residues as mentioned above. This process

is guided by a class of small nucleolar RNAs (snoRNAs)called the box H 1 ACA snoRNAs (Ni et al, 1997). In yeast,the box H 1 ACA snoRNAs form a complex with a numberof nucleolar proteins including Cbf5p, Gar1p, Nhp2p andNop10p, of which Cbf5p is the core component (Henras et al,1998; Lafontaine et al, 1998; Watkins et al, 1998). Theyeast Cbf5p and Drosophila Nop60B/mfl stabilize both theRNA and protein components of the box H 1 ACA complexand have been shown to be pseudouridine synthases. Thisfunctional role would therefore be predicted for dyskerin. Italso suggests that some of the pathology in DC may arisefrom its inability to stabilize other H 1 ACA snoRNAs. Inthis regard, it is of interest that the RNA component (hTR)of telomerase contains the box H 1 ACA structural motif

Fig 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 beenfound 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 1 592 C . G; IVS2 25C . G), a promoter mutation (URR 2142C . G)

and a 2-kb deletion are also marked.

Fig 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 (pseudouridine 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.


Review 775

(Mitchell et al, 1999a). Dyskerin deficiency may thus lead toa problem in stabilizing telomerase, resulting in a secondarydeficiency of telomerase activity. Indeed, Mitchell et al(1999b) have recently demonstrated that DC cell linesfrom two families have a lower level of telomerase RNA,produce lower levels of telomerase activity and have shortertelomeres than matched normal cells. We have studied freshPB samples from 36 DC patients from 22 different families(unpublished observations) and have found that they alsohave significantly shorter telomere lengths than age-matched controls. A deficiency in telomerase could explainsome of the clinical features of DC as compromised telomer-ase function leading to a defect in telomere maintenancemay limit the proliferative capacity of somatic cells (Colgin &Reddel, 1999) in the blood and epithelia. It also shows thatdifferent DKC1 mutations could produce very pleiotropiceffects if different DKC1 mutations have varying effects onthe stabilization of different H 1 ACA snoRNAs, of whichtelemorase is just one representative. Indeed, some featuresof the DC phenotype, such as the severe neurologicalabnormalities seen in the HH variant, are difficult to explainjust on the basis of reduced telomerase activity. Furtherwork is needed to substantiate the functional role ofdyskerin and its interacting proteins and RNAs in the cell.

The skin pigmentation in DC patients can resemble veryclosely, clinically and histologically, the features of chronicgraft-versus-host disease (GVHD) after allogeneic SCT (Linget al, 1985; Ivker et al, 1993). This suggests that althoughthe primary defect in DC is a constitutional geneticabnormality (a putative inherited ribosomapathy; Luzzatto& Karadimitris, 1998), secondary pathology may in part beassociated with immunological abnormalities that areobserved in some DC patients (Solder et al, 1998; Knightet al, 1999a). Indeed, synthesis of abnormal proteins orabnormal rates of synthesis of normal proteins may lead tothe presentation of antigens which, in turn, may inciteautoimmune phenomena. Alternatively, the predicted defectin ribosomal biogenesis may explain the low B-lymphocyteand CD4 counts seen in some patients. The resultingdisturbed CD4/CD8 ratio may lead to altered immuneresponses, including those against host antigens.

Families with affected femalesThree female carriers of X-linked DC had mild clinicalfeatures (single dystrophic nail, a discrete area of pigmenta-tion, mild leucopenia and lumbar scoliosis) and the clinicalphenotype in some female patients is less severe (Knight et al,1998a). This raises the possibility that some of the femalepatients who have hitherto been classified into the auto-somal forms of DC may turn out to have mutations in DKC1.However, to date, we have not found DKC1 mutations in anyfemale cases (unpublished observations). This substantiatesthe case for autosomal DC loci.

Towards haemopoietic gene therapyTreatment for DC patients developing SAA remainsunsatisfactory (Dokal, 1996b) as, even for the small subsetof patients who have compatible sibling donors, the resultsare very poor because of a high incidence of fatal pulmonary

complications. There is therefore a clinical need to developnew treatment strategies for patients developing SAA. AsDC is a single gene recessive disorder and the cells that needto be targeted (haemopoietic stem cells) are accessible, DC isa good candidate for haemopoietic gene therapy. Further-more, there is evidence from fibroblast culture studies andfrom skewed X-chromosome inactivation patterns (XCIPs)in DC carriers that cells transfected with the normal genewould have growth/survival advantage compared with theuncorrected cells. Such an advantage would also bepredicted from the putative role of dyskerin in ribosomebiogenesis.

Haemopoietic studies in DC suggest there is `prematureageing' of the haemopoietic system; there is a reduction inthe proliferative potential of clonogenic haemopoieticprogenitors and a reduced subplating cloning efficiency.Further characterization of DKC1 will facilitate constructionof vectors capable of expressing wild-type DKC1 inhaemopoietic cells. Successful correction of DC haemopoie-tic cells in vitro will form the basis of developing a protocol ofhaemopoietic gene therapy for DC patients. The precisestrategies used for these final studies will depend ontechnical advances in vectors and transfection that arebeing intensively pursued in many laboratories. In addition,information on the regulation of the DKC1 and the avail-ability of a DC mouse model may facilitate the developmentof an efficient haemopoietic gene therapy strategy. Theseexperiments may also have important implications for themanagement of patients with idiopathic AA.

Relationship to idiopathic aplastic anaemiaDyskerin represents the first protein to be identified that isimportant in the primary pathology of AA and wherehomology searches have suggested a possible function forthe protein. To what extent might ribosome biogenesis bedisrupted in idiopathic AA patients? Nucleolar events inwhich dyskerin is involved are currently under intenseinvestigation. Proteins which interact with dyskerin mayturn out to be important targets in the biology not only ofautosomal DC/HH but also of some types of idiopathic AA.

CONCLUSION

DC is a severe multisystem disorder associated withpremature mortality usually due to BM failure/immuno-deficiency for which present forms of treatment are unsatis-factory. The majority of patients are men showing a X-linkedrecessive pattern of inheritance. The identification of theDKC1 gene and its corresponding nucleolar protein,dyskerin, provides an accurate diagnostic test that nowfacilitates early diagnosis, especially in patients presentingwith atypical features such as the Hoyeraal±Hreidarssonsyndrome; patients with unexplained aplastic anaemia orimmunodeficiency and who are below the age of 10 yearsshould be screened for DC. The task over the next few yearsis to determine the function of dyskerin in the normal anddiseased cell. The DKC1 gene also provides a `handle' toidentify the proteins (and their corresponding genes)responsible for autosomal DC/HH and some subtypes of

776 Review


`idiopathic' aplastic anaemia or immunodeficiency. Ashomology searches have suggested that dyskerin has aputative function in ribosome biogenesis, this advance mayshape our understanding of not only the AA associated withDC but also that arising in other patients with hithertocalled `idiopathic' AA. This may, in turn, lead to newtreatment strategies for AA patients who cannot be treatedby haemopoietic stem cell transplantation.

ACKNOWLEDGMENTS

I am indebted to Stuart Knight, Philip Mason and TomVulliamy, whose contributions has been critical over thepast 5 years. I thank my other current (Andy Chase, JohnGoldman, Myrtle Gordon, Samia Hawisa, Jaspal Kaeda, MikeLaffan, Richard Manning, Steve Marley, Irene Roberts,David Stevens) and past (Lucio Luzzatto) colleagues at theHammersmith Hospital, who have contibuted and continueto contribute towards the clinical and scientific aspects ofthe DC project. I am also grateful to all my colleagues fortheir support in establishing the dyskeratosis congenitaregistry (DCR) and to the Wellcome Trust and ActionResearch for their financial support.

Department of Haematology,Imperial College School of Medicine(ICSM), Du Cane Road, London, UK

Inderjeet Dokal

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Keywords: aplastic anaemia, bone marrow failure, chro-mosomal instability, dyskeratosis congenita, Hoyeraal±Hreidarsson syndrome.


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