X-linked recessive dyskeratosis congenita (DKC) is a rare bone-marrow failure disorder linked to Xq28. Hybridization screening with 28 candidate cDNAs resulted in the detection of a 3' deletion in one DKC patient with a cDNA probe (derived from XAP101). Five different missense mutations in five unrelated patients were subsequently identified in XAP101, indicating that it is the gene responsible for X-linked DKC (DKC1). DKC1 is highly conserved across species barriers and is the orthologue of rat NAP57 and Saccharomyces cerevisiae CBF5. The peptide dyskerin contains two TruB pseudouridine (psi) synthase motifs, multiple phosphorylation sites, and a carboxy-terminal lysine-rich repeat domain. By analogy to the function of the known dyskerin orthologues, involvement in the cell cycle and nucleolar function is predicted for the protein.

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

Dyskeratosis congenita is a rare inherited bone marrow-failure syndrome characterized by abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia. More than 80% of patients develop bone-marrow failure, and this is the major cause of premature death. The X-linked form of the disease (MIM 305000) has been shown to be caused by mutations in the DKC1 gene. The gene encodes a 514-amino-acid protein, dyskerin, that is homologous to Saccharomyces cerevisiae Cbf5p and rat Nap57 proteins. By analogy to the homologues in other species, dyskerin is predicted to be a nucleolar protein with a role in both the biogenesis of ribosomes and, in particular, the pseudouridylation of rRNA precursors. We have determined the genomic structure of the DKC1 gene; it consists of 15 exons spanning a region of 15 kb. This has enabled us to screen for mutations in the genomic DNA, by using SSCP analysis. Mutations were detected in 21 of 37 additional families with dyskeratosis congenita that were analyzed. These mutations consisted of 11 different single-nucleotide substitutions, which resulted in 10 missense mutations and 1 putative splicing mutation within an intron. The missense change A353V was observed in 10 different families and was shown to be a recurring de novo event. Two polymorphisms were also detected, one of which resulted in the insertion of an additional lysine in the carboxy-terminal polylysine domain. It is apparent that X-linked dyskeratosis congenita is predominantly caused by missense mutations; the precise effect on the function of dyskerin remains to be determined.

Familial incontinentia pigmenti (IP; MIM 308310) is a genodermatosis that segregates as an X-linked dominant disorder and is usually lethal prenatally in males. In affected females it causes highly variable abnormalities of the skin, hair, nails, teeth, eyes and central nervous system. The prominent skin signs occur in four classic cutaneous stages: perinatal inflammatory vesicles, verrucous patches, a distinctive pattern of hyperpigmentation and dermal scarring. Cells expressing the mutated X chromosome are eliminated selectively around the time of birth, so females with IP exhibit extremely skewed X-inactivation. The reasons for cell death in females and in utero lethality in males are unknown. The locus for IP has been linked genetically to the factor VIII gene in Xq28 (ref. 3). The gene for NEMO (NF-kappaB essential modulator)/IKKgamma (IkappaB kinase-gamma) has been mapped to a position 200 kilobases proximal to the factor VIII locus. NEMO is required for the activation of the transcription factor NF-kappaB and is therefore central to many immune, inflammatory and apoptotic pathways. Here we show that most cases of IP are due to mutations of this locus and that a new genomic rearrangement accounts for 80% of new mutations. As a consequence, NF-kappaB activation is defective in IP cells.

Mutations in the DKC1 gene are responsible for causing the bone marrow failure syndrome, dyskeratosis congenita (DKC; OMIM 305000). The majority of mutations identified to date are missense mutations and are clustered in exons 3, 4 and 11. It is predicted that the corresponding protein dyskerin is a nucleolar phosphoprotein which functions in both pseudo-uridylation and cleavage of precursor rRNA. Dyskerin contains multiple putative nuclear localization signals (NLSs) at the N-terminus (KKHKKKKERKS) and C-terminus [KRKR(X)(17)KKEKKKSKKDKKAK(X)(17)-KKKKKKKKAKEVELVSE]. By fusing dyskerin with the enhanced green fluorescent protein (EGFP) and by following a time course of expression in mammalian cell lines, we showed that full-length dyskerin initially localizes to the nucleoplasm and subsequently accumulates in the nucleoli. A co-localization to the coiled bodies was observed in some cells where dyskerin-EGFP had translocated to the nucleoli. Analysis of a series of mutant constructs indicated that whereas the most C-terminal lysine-rich clusters [KKEKKKS-KKDKKAK(X)(17)KKKKKKKKAKEVELVSE] influence the rate of nucleoplasmic and nucleolar accumulation, the KRKR sequence is primarily responsible for the nuclear import. Nucleolar localization was maintained when either the N- or C-terminal motifs were mutated, but not when all NLSs were removed. We conclude that the intranuclear localization of dyskerin is accomplished by the synergistic effect of a number of NLSs and that the nucleolar localization signals are contained within the NLSs. Further, examination of dyskerin-EGFP fusions mimicking mutations detected in patients indicated that the intracellular mislocalization of dyskerin is unlikely to cause DKC.

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