DKC1 Research Articles

Absence of DKC1 exon 3 mutation in common human cancers

Dyskeratosis congenita is a genetic disease caused by point muations in the DKC1 gene [1]. Patients with dyskeratosis congenita show bone marrow failure syndrome characterized by abnormal skin pigmentation and mucosal leukoplakia [2]. Of note, a subset of patients with dyskeratosis congenita developed malignant tumors of various histologic origins [2]. Also, DKC1 mutant mice were highly susceptible to tumor development, and the most common tumors were lung and breast tumors [3]. These observations suggested that DKC1 may be a tumor suppressor gene. DKC1 protein normally binds with small nucleolar RNAs and the RNA components of telomerase, and these functions are impaired in the cells of the DKC1 mutant mice [3]. In the individuals with dyskeratosis congenita, DKC1 gene mutation is detected usually in the exon 3, strongly suggesting that these mutations may related with the disease phenotypes [1,4,5]. It could be hypothesized that increased incidence of malignant tumors in the dyskeratosis congenita patients might be related to the DKC1 mutations. It is of interest to see whether the DKC1 gene is somatically mutated in human cancers. However, to date, the data on the somatic mutations of DKC1 in human sporadic cancers is lacking. The aim of this study was to see whether common human sporadic cancers harbor DKC1 mutation. We have analyzed methacarn-fixed tissues of 140 gastric carcinomas, 104 colorectal carcinomas, 94 breast ductal carcinomas, 100 non-small cell caners and 69 hepatocellular carcinomas. All of the patients of the cancers were Asians (Koreans). The gastric carcinomas consisted of 60 diffuse-type, 49 intestinal-type and 31 mixed-type gastric adenocarcinomas by Lauren’s classification, and 25 early and 115 advanced gastric carcinomas according to the depth of invasion. The colorectal carcinomas originated from cecum (n /2), ascending colon (n /19), transverse colon (n /6), descending colon (n /4), sigmoid colon (n /28) and rectum (n /45). The breast carcinomas consisted of 15 intraductal and 79 invasive ductal carcinomas. The non-small cell lung cancer samples consisted of 50 adenocarcinomas, 47 squamous cell carcinomas, 1 adenosquamous carcinomas and 2 large cell carcinomas. The hepatocellular carcinomas consisted of Edmondson grade I (n /8), grade II (n /30) and grade III (n / 31) according to Edmondson and Steiner’s criteria [6]. Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator, as described previously [7]. DNA extraction was performed by a modified single-step DNA extraction method [7]. Because the most common site of the germline mutations of DKC1 was reported to be the exon 3 [1,4,5], we analyzed the exon 3 in this study. Genomic DNA each from tumor cells and corresponding normal cells were amplified with a primer pair covering the DNA sequences in the exon 3. For the detection of DKC1 mutations, we analyzed the exon by

Sex Hormones Up-Regulate Telomerase Activity of Normal Human Hematopoietic Cells and Restore Telomerase Activity in Carriers of Telomerase Complex Mutations.

Mutations in components of the telomerase complex (TERT and TERC) are risk factors for acquired aplastic anemia. Telomerase is a reverse transcriptase responsible for the maintenance of telomeres, repetitive nucleotide sequences capping the chromosome ends. Patients with marrow failure with TERT or TERC mutations have low telomerase activity, short telomere length in leukocytes, reduced hematopoietic function, and they do not respond adequately to immunosuppressive therapy. The constitutional aplastic anemia dyskeratosis congenita is also caused by mutations in TERC (autosomal recessive type) or in an additional component of the telomerase complex, dyskerin, encoded by the DKC1 gene (X-linked type). The majority of patients with dyskeratosis congenita and some patients with acquired aplastic anemia benefit from androgen therapy, but the mechanism by which this hormone stimulates hematopoiesis is not well understood. We investigated whether sex hormones stimulated telomerase activity in hematopoietic tissue. Peripheral blood lymphocytes from either healthy controls or mutation carriers were cultured with phytohemagglutinin and interleukin-2 for 1–7 days in the presence of methyltrienolone, estradiol, hydroxycortisone, and/or tamoxifen. Telomerase enzymatic activity was measured using a fluorescent telomere-repeating amplification protocol (TRAP), and TERT mRNA expression was assayed by real-time PCR. Androgens and estradiol up-regulated telomerase activity in lymphocytes from normal subjects in a dose-dependent fashion (controls, 124±24 total product generated [TGP] units per reaction; methyltrienolone 1 μM, 242±16 TGP units/reaction; methyltrienolone 10 μM, 757±87 TGP units/reaction; P<0.01). Increased telomerase activity correlated to higher TERT mRNA levels (P<0.05). Steroidal effects on telomerase activity were neutralized by tamoxifen, an estrogen antagonist. TERT mutation carriers had lower telomerase activity compared to controls (P<0.05), but their enzymatic function was restored to normal levels when cultured with methyltrienolone. As a control, hydrocortisone at 1 μM inhibited telomerase activity without influencing cell cycle or inducing apoptosis. We also investigated the effects of androgen on hematopoietic progenitor cells. Bone marrow CD34+ cells from normal subjects were immunomagnetically separated and cultured for eight days in StemSpan supplemented with Flt-3 ligand, stem cell factor, and interleukins 3 and 6, in the presence methyltrienolone. Up-regulation of telomerase activity was observed in CD34+ cells cultured with methyltrienolone (P<0.05), the increase correlated with greater cell numbers, a higher proportion of CD34+ cells after eight days of culture (from 10% to 15% at 10 μM methyltrienolone and to 19% at 100 μM), without changes in cell cycle or cell differentiation, based on CD45, CD71, and CD33 expression. These results indicate that sex hormones stimulate TERT expression via the estrogen receptors, leading to increased telomerase activity in hematopoietic cells and increased proliferation of hematopoietic progenitors. Androgens were able to restore telomerase activity to normal levels in TERT mutation carriers. Sex hormone effects on telomerase in normal cells may have implications for the normal aging process and in hematological malignancy (leukemic cells have high telomerase activity), and our results suggest that androgens might be useful in the treatment of patients bearing telomerase complex mutations.

Evidence for T-Cell Oligoclonal Expansion in Aplastic Anemia Associated with Telomerase Complex Mutations: Pathophysiological and Clinical Implications.

Aplastic anemia is characterized by peripheral blood pancytopenia and a hypocellular bone marrow. In the majority of patients, bone marrow destruction is immune-mediated by a type-1 T cell response, dominated by oligoclonal expansion of CD8+ cells and targeting hematopoietic stem cells. Some patients with acquired aplastic anemia are heterozygous for mutations in genes encoding the major components of the telomerase complex, telomerase reverse transcriptase (encoded by TERT; Yamaguchi et al., N Engl J Med 2005; 352:1413) and the RNA component (encoded by TERC; Fogarty et al., Lancet 2003; 362:1628). These mutations lead to shortened telomeres of leukocytes, low telomerase activity and reduced hematopoietic function. Relatives carrying the same mutations also have short telomeres and reduced hematopoietic function, but they do not inevitably develop overt marrow failure. In contrast, dyskeratosis congenita, a constitutional type of marrow failure, is caused by mutations in TERC (autosomal dominant type) or in the DKC1 gene, which encodes an additional component of the telomerase complex, dyskerin. In addition to marrow failure, patients with dyskeratosis congenita also often have physical anomalies, such as leukoplakia, nail dystrophy, and hyperpigmentation as well as hepatic or pulmonary fibrosis. In dyskeratosis congenita, family members bearing the genetic mutation present variable degrees of physical abnormalities or organ damage. However, in patients with acquired aplastic anemia carrying telomerase mutations without physical anomalies, it is unclear whether these mutations are sufficient for the development of marrow failure. We hypothesized that telomerase complex mutations reduce the size of the hematopoietic stem cell compartment and affect its regenerative capacity, making carriers more vulnerable to environmental insults or autoimmune damage. We analyzed the distribution of the T-cell repertoire (T-cell receptor [TCR] Vβ subfamily) and expansion of particular Vβ subsets by flow cytometry in peripheral blood of six aplastic anemia patients carrying telomerase complex mutations (five with TERT mutations, one with a TERC mutation), two patients with Fanconi anemia, and 15 healthy subjects. The expression of 22 Vβ subfamilies were evaluated in CD8+CD28− cells, and expansion was defined when the percentage of a given Vβ subfamily was above two standard deviations based on the control group (Risitano et al. Lancet 2004; 364:355). We also evaluated interferon-γ levels in serum of nine telomerase mutant patients and 10 controls by ELISA. Expanded Vβ subsets were observed in all six aplastic patients carrying telomerase complex mutations analyzed. Five patients (with TERT mutations) had two clones expanded and one (with TERC mutation) showed three overrepresented clones. The Vβ subsets 9, 13.6, 17, and 20 were expanded in more than one patient. Oligoclonal Vβ expansion was not observed in Fanconi anemia. Telomerase mutant patients also had significantly increased interferon-γ serum levels in comparison to controls (27 pg/mL; range, 0–95 vs. 7.6, 0–16, respectively; P<0.02). These results indicate that an immune process targeting hematopoiesis may operate in patients carrying telomerase complex gene mutations. Limited responsed to immunosuppression may reflect the poor hematopoietic reserve rather than a nonimmunologic mechanism of marrow destruction.

Identification of DKC1 gene mutations in Japanese patients with X‐linked dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited multisystem disorder characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leucoplakia. X-linked recessive inheritances are recognized in approximately 40% of the patients. DKC1 has been identified as the gene responsible for X-linked DC, and genetic analyses have been performed in a worldwide study. Here, we performed genetic analysis of five Japanese patients with presumed X-linked DC, and identified four mutations in the DKC1 gene, including two novel missense mutations (Q31K and T357A). Such genetic analysis is useful for the definite diagnosis and genetic counselling of patients.

Mutations inTERT,the Gene for Telomerase Reverse Transcriptase, in Aplastic Anemia

Mutations in TERC, the gene for the RNA component of telomerase, cause short telomeres in congenital aplastic anemia and in some cases of apparently acquired hematopoietic failure. We investigated whether mutations in genes for other components of telomerase also occur in aplastic anemia. We screened blood or marrow cells from 124 patients with apparently acquired aplastic anemia and 282 control subjects for sequence variations in the TERT, DKC1, NHP2, and NOP10 genes; an additional 81 patients and 246 controls were examined for genetic variations in TERT. Telomere lengths and the telomerase activity of peripheral-blood leukocytes were evaluated in patients carrying genetic variants. Identified mutations were transfected into telomerase-deficient cell lines to examine their effects and their mechanism of action on telomerase function. Five heterozygous, nonsynonymous mutations (which cause an amino acid change in the corresponding protein) were identified in TERT, the gene for the telomerase reverse transcriptase catalytic enzyme, among seven unrelated patients. Leukocytes from these patients had short telomeres and low telomerase enzymatic activity. In three of these patients, the mutation was also detected in buccal mucosa cells. Family members carrying the mutations also had short telomeres and reduced telomerase activity but no evident hematologic abnormality. The results of coexpression of wild-type TERT and TERT with aplastic anemia-associated mutations in a telomerase-deficient cell line suggested that haploinsufficiency was the mechanism of telomere shortening due to TERT mutations. Heterozygous mutations in the TERT gene impair telomerase activity by haploinsufficiency and may be risk factors for marrow failure.

A Dyskeratosis Congenita (DKC) Patient Treated with Growth Hormone

Dyskeratosis congenita (DKC) is an inherited disease characterized by the triad of mucosal leukoplakia, abnormal skin pigmentation and nail dystrophy, and it usually appears between the ages of 5 and 10 yr. Reports show that 19.5% of DKC patients have short stature (1). A major complication of DKC is progressive development of pancytopenia and there is also an increased incidence of malignancies. The X-linked form accounts for the majority of cases and is caused by mutations in the DKC1 gene. We report a 9 yr old boy, who presented with the triad of DKC, a mutation in the DKC1 gene and short stature, who was treated with human recombinant growth hormone (hGH). This is the first report of a DKC patient treated with hGH.

Disqueratosis congénita ligada al cromosoma X

Dyskeratosis congenita is a rare genetic disorder with approximately 180 cases reported in the literature. Objective: To present the case of a boy, in whom the diagnosis was clinically suspected and later confirmed by molecular analysis of the Dyskerin gene. Clinical Case: A school age boy with a history of anemia resistant to treatment, and severe bacterial infections, who presented with reticulated pigmentation of the skin, nail dystrophy, oral leukoplakia and progressive pancitopenia. Molecular analysis revealed a mutation in the DKC1 gene, that results in an aminoacid change from alanine to valine. Conclusion: Dyskeratosis congenita is a rare inherited disease characterised by cutaneous reticulated hyperpigmentation, nail dystrophy, oral leukoplakia and progressive pancytopenia. The mutation detected in this patient is an important cause of dyskeratosis congenita worldwide.

Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein-RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis.

Enhanced telomere shortening in transformed lymphoblasts from patients with X linked dyskeratosis

Aim: Dyskeratosis congenita (DC) is characterised by the failure of those tissues that are rapidly dividing in the adult, particularly the skin, mucosae, and haemopoietic system. The X linked form...

Targeted disruption of Dkc1, the gene mutated in X-linked dyskeratosis congenita, causes embryonic lethality in mice

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome associated with increased cancer susceptibility. The X-linked form is due to mutations in the DKC1 gene encoding dyskerin, a nucleolar protein predicted to be involved in rRNA processing and associated with the telomerase complex. Available evidence suggests the pathology of DC is due to telomerase defects. We have used the inducible Cre/loxP system to produce deletions in the murine Dkc1 gene in early embryogenesis. A large deletion lacking exons 12-15 and a small deletion lacking only the last exon, were produced. We found both deletions showed a parent-of-origin effect with 100% embryonic lethality when the mutation occurred on the maternal Dkc1. Embryonic analysis at day E7.5 and E9.5 showed no male embryos carrying either deletion whereas females with maternally derived deletions died around day E9.5, with degeneration of the extra embryonic tissue, in which the paternal X-chromosome is inactivated. Female mice carrying the deletion in the paternally derived Dkc1 show extreme skewing of X-inactivation with the wild type X-chromosome active in all cells. Since mice with no telomerase are viable in the first generations the lethality we observe is unlikely to be due to the effects of mutated dyskerin on telomerase activity.

Basal transcription activity of the dyskeratosis congenita gene is mediated by Sp1 and Sp3 and a patient mutation in a Sp1 binding site is associated with decreased promoter activity

The multisystem disorder dyskeratosis congenita (DKC) is caused by mutations in the DKC1 gene. The protein dyskerin is a component of the box H+ACA small nucleolar RNAs (snoRNAs) and is also functionally associated with the RNA component of the human telomerase. The majority of mutations are missense mutations, although single examples of non-coding mutations have been described. One of these is a point mutation in a putative Sp1 binding site in the 5′-upstream region of the DKC1 gene which presumably represents the promoter region of the gene. In this report, we compare the promoter sequences of both the human and mouse genes and provide a first functional characterisation of the human DKC1 promoter. This includes a characterisation of the disease-associated implications caused by the mutation identified in one patient. By reporter gene analysis, functional regions of the DKC1 promoter were delineated. The core promoter region critical for basal level of transcription was found to lie at −10 to −180. Bandshift- and supershift experiments clearly demonstrated a mutual binding of transcription factors Sp1 and Sp3 to two of five putative GC-box/Sp1-binding sites located within the core promoter region. An additional GC-box interacts only with the Sp1 transcription factor. Further, we provide evidence that the DKC1 mutation in one of the Sp1 binding sites results in reduced promoter activity.

A NOVEL MISSENSE MUTATION IN THE DKC1 GENE IN A JAPANESE FAMILY WITH X-LINKED DYSKERATOSIS CONGENITA

The authors report 2 male patients with dyskeratosis congenita (DC) in a Japanese kindred. Sequencing of the complementary DNA of the dyskerin gene (DKC1) revealed a T-to-C transition at nucleotide 1285 in exon 12 that resulted in a novel missense mutation L398P. Despite harboring the same mutation in the DKC1 gene, one patient had significantly milder hematological symptoms than the other, indicating that there may be other factors that determine the severity of DC.

Increased Mortality Rate and Not Impaired Ribosomal Biogenesis is Responsible for Proliferative Defect in Dyskeratosis Congenita Cell Lines

X-linked dyskeratosis congenita is a rare inherited disorder mainly characterized by progressive changes in proliferating epidermal, mucosal, and bone marrow tissues that commonly emerge after 10 y of life. It is caused by mutations of the DKC1 gene, which codes for dyskerin, a protein that may play a role in ribosomal biogenesis. In order to verify whether the defects of proliferating tissues observed in dyskeratosis congenita are due to an altered ribosome synthesis, we studied ribosomal biogenesis in relation to cell proliferation in two lymphoblastoid cell lines from dyskeratosis congenita patients and in one control line. We observed that in the dyskeratosis congenita cell lines the rRNA transcription and maturation and proliferative capability remained unimpaired. Increasing the number of cell cycles, however, leads to a steep rise in the apoptotic fraction of dyskeratosis congenita cells, which is not observed in controls. These findings demonstrate that whereas dyskeratosis congenita cell lines do not display proliferation defects, they do show progressively increasing levels of apoptosis in relation to the number of cell divisions. This concept is consistent with (i) the delayed onset of dyskeratosis congenita proliferating-tissue defects, which do not emerge during embrional development as would be expected with ribosomal biogenesis alterations, and (ii) with the increasing severity of the proliferating-tissue defects over time.

Cloning and characterization of Arabidopsis thaliana AtNAP57--a homologue of yeast pseudouridine synthase Cbf5p.

Rat Nap57 and its yeast homologue Cbf5p are pseudouridine synthases involved in rRNA biogenesis, localized in the nucleolus. These proteins, together with H/ACA class of snoRNAs compose snoRNP particles, in which snoRNA guides the synthase to direct site-specific pseudouridylation of rRNA. In this paper we present an Arabidopsis thaliana protein that is highly homologous to Cbf5p (72% identity and 85% homology) and NAP57 (67% identity and 81% homology). Moreover, the plant protein has conserved structural motifs that are characteristic features of pseudouridine synthases of the TruB class. We have named the cloned and characterized protein AtNAP57 (Arabidopsis thaliana homologue of NAP57). AtNAP57 is a 565 amino-acid protein and its calculated molecular mass is 63 kDa. The protein is encoded by a single copy gene located on chromosome 3 of the A. thaliana genome. Interestingly, the AtNAP57 gene does not contain any introns. Mutations in the human DKC1 gene encoding dyskerin (human homologue of yeast Cbf5p and rat NAP57) cause dyskeratosis congenita a rare inherited bone marrow failure syndrome characterized by abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia.

Identification of novel DKC1 mutations in patients with dyskeratosis congenita: implications for pathophysiology and diagnosis.

Dyskeratosis congenita (DC) is characterised by the failure of those tissues that are rapidly dividing in the adult, particularly the skin and haemopoietic system. The X-linked form of the disease is caused by mutations in the DKC1 gene. To date the only DKC1 mutations detected result in alterations in the amino acid sequence of dyskerin. Dyskerin is the catalytic subunit of the H+ACA box small nucleolar RNA particles responsible for the site-specific pseudouridination of rRNA and in humans is also a component of the telomerase complex. In order to further characterise the disease at the molecular level, male DC patients from 25 families were screened for mutations in the DKC1 gene. Sequence variations were detected in 10 of these families. In five families, previously identified mutations were detected. Of the five novel sequence changes, three were coding changes: R158 W, S280R and P384L. A fourth sequence change was detected in the 5'-flanking region that disrupts a putative Spl transcription factor binding site. An intronic change was also detected that resulted in the partial incorporation of a portion of intron 1 into the mRNA. The identification of this mutation highlights the importance of screening for mutations that cause the partial aberrant splicing of mRNA. This is the first report of DKC1 mutations that are predicted to affect the level of expression of dyskerin. This suggests that a decrease in the amount of the normal protein may cause the disease.

Gene Structure and Expression of the Mouse Dyskeratosis Congenita Gene, Dkc1

Mutations in the DKC1 gene are responsible for causing X-linked recessive dyskeratosis congenita (DKC) and a more severe allelic variant of the disease, Hoyeraal–Hreidarsson syndrome. Both diseases are characterized by progressive and fatal bone marrow failure. The nucleolar protein dyskerin is the pseudouridine synthase component of the box H+ACA snoRNAs and also interacts with the RNA component (human telomerase, hTR) of the telomerase complex. Dyskerin is therefore thought to function in the processing of pre-rRNA and of the hTR, strengthening the notion that the underlying mechanism of DKC is a premature senescence of cells, especially of the rapidly dividing epithelial and hemopoietic cells. To examine the functions of dyskerin in vivo, it will be necessary to generate mouse models. As a first step, we here provide the genomic structure of the mouse Dkc1 gene and expression analysis of the transcript. Northern hybridizations revealed the tissue-specific expression of an alternative 4.5-kb transcript, in addition to the ubiquitous 2.6-kb transcript. RNA in situ hybridizations on day 10.5–18.5 postconception embryos showed a ubiquitous expression of Dkc1 with a notably higher level of expression confined to the epithelial tissues. In addition, higher level Dkc1 expression was confined to embryonic neural tissues as well as to specific neurons in the cerebellum (Purkinje cells) and the olfactory bulb (mitral cells) of the adult brain. In adult testis, elevated expression was limited to the Leydig cells. The results indicate that some of the pertinent functions of dyskerin may be more tissue-specific than previously thought and are not limited to rapidly dividing cells.

Molecular medicine and bone marrow failure syndromes

Molecular medicine and bone marrow failure syndromes

Overlap of dyskeratosis congenita with the Hoyeraal-Hreidarsson syndrome

X-linked dyskeratosis congenita (DKC) is characterized by mucosal leukoplakia and ulcerations, skin abnormalities, nail dystrophy, and pancytopenia. Hoyeraal-Hreidarsson syndrome (HHS) includes intrauterine growth retardation, microcephaly, mental retardation, cerebellar malformation, and pancytopenia. A patient with striking features of both HHS and DKC has a de novo mutation in the DKC1 gene, known to be responsible for DKC. HHS may be a severe form of DKC, in which affected individuals die before characteristic mucocutaneous features develop. (J Pediatr 2000;136:390-3)

Dyskerin localizes to the nucleolus and its mislocalization is unlikely to play a role in the pathogenesis of dyskeratosis congenita.

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.

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

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