One novel and two recurrent missense DKC1 mutations in patients with dyskeratosis congenita (DKC).

X-linked dyskeratosis congenita (DKC) is a progressive multisystem disorder most severely affecting tissues with a high cellular turnover such as skin, mucous membranes, and blood. Most patients die of bone marrow failure, although the chances of succumbing to various types of cancer and pulmonary disease are also high. DKC is caused predominantly by missense mutations in the DKC1 gene linked to Xq28. Some of the clinical features are reminiscent of premature ageing and this agrees with recent indications that DKC could be a telomere maintenance disorder. There is considerable variability in the type, severity, and age at onset of the various anomalies. Recognition of this has increased with the finding that patients with Hoyeraal-Hreidarsson syndrome (HHS) who exhibit severe neurological problems in addition to early-onset pancytopenia, also bear mutations in the DKC1 gene. For these reasons, and compounded by the range of mutations, phenotype-genotype correlations and accurate assessments of prognosis have not been possible. To complement the present data, we here report on three new cases of DKC and their mutations. One is a novel mutation in the exon 3 (K43E). The other two represent a frequently recurring mutation in exon 11 (A353V) and a less frequently recurring mutation in the exon 3 (T49M).

RAI1 is a novel polyglutamine encoding gene that is deleted in Smith-Magenis syndrome patients.

The human chromosomal band 17p11.2 is a genetically unstable interval. It has been shown to be deleted in patients suffering from Smith-Magenis syndrome. Previous efforts of physical and transcriptional mapping in 17p11.2 and subsequent genomic sequencing of the candidate interval allowed the identification of new genes that might be responsible for the Smith-Magenis syndrome. In this report, one of these genes named RAI1, the human homologue of the mouse Rai1 gene, has been investigated for its contribution to the syndrome. Expression analysis on different human adult and fetal tissues has shown the existence of at least three splice variants. Moreover, the most interesting feature of the gene is the presence of a polymorphic CAG repeat coding for a polyglutamine stretch in the amino terminal domain of the protein.

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.

Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti. The International Incontinentia Pigmenti (IP) Consortium.

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.

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.

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.

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.

Dyskeratosis congenita caused by a 3' deletion: germline and somatic mosaicism in a female carrier.

X-linked dyskeratosis congenita (DC) is a bone marrow failure syndrome caused by mutations in the DKC1 gene located at Xq28. By 20 years of age, most affected boys develop bone marrow failure, whereas female carriers show a skewed pattern of X-chromosome inactivation. The gene product, dyskerin, is homologous to a yeast protein involved in ribosomal RNA biogenesis, providing a unique insight into a cause of aplastic anemia. Whereas most causative mutations are single amino acid substitutions, and nonsense or frameshift mutations have not been observed, we present here a case of DC caused by a 2-kb deletion that removes the last exon of the gene. Normal levels of mRNA are produced from the deleted gene, with the transcripts using a cryptic polyadenylation site in the antisense strand of the adjacent MPP1 gene, normally located 1 kb downstream of DKC1 in a tail to tail orientation. The predicted truncated protein lacks a lysine-rich peptide that is less conserved than the rest of the dyskerin molecule and is dispensable in yeast, supporting the contention that it may retain some activity and that null mutations at this locus may be lethal. The affected boy had an unaffected brother with the same haplotype around the DKC1 gene and a sister who was heterozygous for the deletion. We conclude therefore that the mother must be a germline mosaic with respect to this deletion. Investigation of her blood cells and other somatic tissues showed that a small proportion of these cells also carried the deletion, making her a somatic mosaic and indicating that the deletion took place early in development.

X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene.

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.

Transcription mapping in a medulloblastoma breakpoint interval and Smith-Magenis syndrome candidate region: identification of 53 transcriptional units and new candidate genes.

The chromosomal band 17p11.2 is associated with a number of neurological disorders and malignant diseases. This region is also characterized by the presence of complex repeat elements that are probably responsible for the frequent occurrence of interstitial deletions, duplications, and isochromosome formation. In the course of the molecular analysis of this interval, an integrated map with YACs, PACs, and cosmids covering approximately 6 Mb was established. Focusing on the 1.4-Mb interval containing the Smith-Magenis syndrome critical region and the breakpoint region for medulloblastomas, we constructed a detailed transcript map between the marker PS2 and the proximal CMT1A repeat. FISH analysis of the PACs allowed determination of the position of the transcripts with respect to the SMS critical region and the presumptive chromosomal breakpoint in medulloblastomas. One PAC (G21100) provided evidence for the presence of a novel complex repeat unit, indicating that there are at least three independent repeat elements within 2 Mb. Five genes were mapped to clone G21100 and are likely to form part of this novel complex sequence repeat. In summary, 53 new transcripts were isolated by using cDNA selection and exon trapping. This included 8 known but previously unmapped genes and 45 novel transcripts. The expression profile of 21 transcripts was determined by RT-PCR. Based on their homologies to known genes or proteins, some of the novel genes are considered candidate genes either for malignant diseases or for the Smith-Magenis syndrome.

1.4 Mb candidate gene region for X linked dyskeratosis congenita defined by combined haplotype and X chromosome inactivation analysis.

Dyskeratosis congenita (DC) is a rare inherited disorder characterised by the early onset of reticulate skin pigmentation, nail dystrophy, and mucosal leucoplakia. In over 80% of cases bone marrow failure develops and this is the main cause of early mortality. The DC1 gene responsible for the X linked form (MIM 305000) of dyskeratosis congenita has been mapped to Xq28. In order to narrow the candidate gene region, genetic linkage analysis was performed in eight X linked pedigrees using a set of markers spanning Xq28. A maximum lod score of 5.31 with no recombinations was achieved with marker DXS1073. Two recombination events were identified; one of these uses X chromosome inactivation pattern analysis to determine carrier status and haplotype analysis to fine map the recombination breakpoint. The fine mapping of these recombination events has enabled the candidate gene region for X linked dyskeratosis congenita to be defined as the 1.4 Mb interval between Xq3274 and DXS1108.

Genomic organization of a 225-kb region in Xq28 containing the gene for X-linked myotubular myopathy (MTM1) and a related gene (MTMR1).

MTM1 is responsible for X-linked recessive myotubular myopathy, which is a congenital muscle disorder linked to Xq28. MTM1 is highly conserved from yeast to humans. A number of related genes also exist. The MTM1 gene family contains a consensus sequence consisting of the active enzyme site of protein tyrosine phosphatases (PTPs), suggesting that they belong to a new family of PTPs. Database searches revealed homology of myotubularin and all related peptides to the cisplatin resistance-associated alpha protein, which implicates an as yet unknown function. In addition, homology to the Sbf1 protein (SET binding factor 1), involved in the oncogenic transformation of fibroblasts and differentiation of myoblasts, was also evident. We describe 225 kb of genomic sequence containing MTM1 and the related gene, MTMR1, which lies 20 kb distal to MTM1. Although there is only moderate conservation of the exons, the striking similarity in the gene structures indicates that these two genes arose by duplication. Calculations suggest that this event occurred early in evolution long before separation of the human and mouse lineages. So far, mutations have been identified in the coding sequence of only 65% of the patients analyzed, indicating that the remaining mutations may lie in noncoding regions of MTM1 or possibly in MTMR1. Knowledge of the genomic sequence will facilitate mutation analyses of the coding and noncoding sequences of MTM1 and MTMR1.

X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions.

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.

Genomic structure of a novel chloride channel gene, CLIC2, in Xq28.

A transcript map was previously constructed in the 1200-kb telomeric region of Xq28. One of the cDNAs, XAP121, displayed homology to a p64 bovine chloride channel and to a human chloride channel (p64CLCP, NCC27) at both the nucleotide and the peptide levels. In addition, all three sequences exhibited homologies to numerous ESTs derived from human, mouse, rat and pig. While the human NCC27 and XAP121 homologs encode small peptides of 241 and 243 amino acids, respectively, the bovine peptide has a length of 437 amino acids. This suggests that the human genes represent a novel and separate class of small chloride channels. Unlike other chloride channels, the NCC27 peptide was recently shown to localize intracellularly in the cytoplasm and nucleus. The NCC27 and XAP121 genes have thus been designated CLIC1 and CLIC2 for chloride intracellular channel genes 1 and 2, respectively. Since a direct association exists between a number of human chloride channel genes and a range of hereditary diseases, CLIC2 possibly represents a candidate for one of the many diseases linked to Xq28. To facilitate defined mutation analyses, we determined the genomic structure of the CLIC2 gene.

Genomic structure of a novel LIM domain gene (ZNF185) in Xq28 and comparisons with the orthologous murine transcript.

Construction of a transcript map in the DXS52 region in Xq28 had previously led to the isolation of a cDNA with a LIM zinc finger domain in the carboxyl terminus. In parallel, the orthologous murine transcript was isolated from the syntenic region. The human and mouse cDNAs have been designated ZNF185 and Zfp185, respectively. By integrating the cDNA sequence with the cosmid-derived genomic sequence the exon-intron structure of the 3' end of the ZNF185 gene was resolved. Comparative sequence analyses of the human genomic sequence with the full-length murine cDNA facilitated prediction of the 5' end of the gene. The selective expression of three transcripts corresponding to the ZNF185 gene and a related gene was shown by Northern and Southern blots. In situ hybridizations revealed a nonubiquitous and stage-specific expression of Zfp185, especially in differentiating connective tissue. Since LIM proteins regulate cellular proliferation and/or differentiation by diverse mechanisms, and some have directly been associated with disease, conceivably ZNF185 may represent a candidate for a disease-causing gene linked to Xq28. Knowledge of the genomic structure will permit detailed mutation analyses.

Transcriptional analysis of the candidate region for incontinentia pigmenti (IP2) in Xq28.

The hereditary form of incontinentia pigmenti (IP2) is a rare disorder characterized by abnormalities of the tissues and organs derived from the ectoderm and neuroectoderm and has been linked to Xq28 distal to the factor VIII gene (F8C). Four YAC clones covering the 1.1-Mb candidate region at the telomere of Xq28 were subjected to direct cDNA selection and Alu long-range PCR. The products of both methods were subsequently used to isolate 154 cosmid clones that were assembled into five cosmid contigs. This first-generation cosmid map covered the region almost entirely and was used as a basis for constructing a transcript map that was in turn integrated with the physical YAC and cosmid maps. To isolate specifically coding sequences, exon trapping and cDNA selection methods were combined. Exon trapping was carried out on YAC Alu-PCR products, YAC Alu long-range PCR products, and on pools of cosmids. The region-specific enriched cDNA library was then screened by using the exon trap products as complex probes. To ensure a more complete analysis, the products from cDNA selection experiments were also used to screen conventional oligo(dT) primed cDNA libraries. Twenty overlapping cDNA contigs were assembled and computer analyses were performed to identify EST hits, open reading frames, protein motifs, and protein sequence homologies. Five of the cDNA contigs corresponded to known sequences such as the factor VIII, c6.1A, and c6.1B. genes, and both distal copies of the factor VIII intron 22 repeat sequence. Expression patterns of the 15 new cDNA contigs were analyzed by Northern blot and RT-PCR studies and these data were integrated with expression data obtained from known EST sequences. Although a more detailed analysis of this 1.1-Mb region with respect to the structure and function of the genes will only ultimately be possible by a global sequencing approach, an analysis of all novel transcripts as candidate genes for incontinentia pigmenti is already in progress.

Transcription mapping in a 700-kb region around the DXS52 locus in Xq28: isolation of six novel transcripts and a novel ATPase isoform (hPMCA5).

The chromosomal band Xq28 has been a focus of interest in human genetics because > 20 hereditary diseases have been mapped to this region. However, about two-thirds of the disease genes remain uncloned. The region around the polymorphic DXS52 locus (ST14) within Xq28 lies in the candidate regions for several as-yet-uncloned disease genes. So far, only four melanoma antigen genes (MAGE) and the human biglycan (BGN) gene, have been mapped within the 700-kb stretch around DXS52, suggesting that more genes may reside in this region. By combining exon trapping and direct cDNA selection methods, we sought to identify novel transcripts around the DXS52 locus. In addition to recovering the MAGE and BGN genes, we isolated and mapped six putative novel genes (XAP103-XAP108), the caltractin gene, and a gene encoding a novel Ca(2+)-transporting ATPase isoform (hPMCA5). The newly isolated sequences were considered as representing parts of putative genes if they contained at least one unique exon-trap product and/or at least one expressed sequence tag (EST) from sequence data bases and if, in addition, they showed evidence of expressed RT-OCT and/or Northern blot analysis. Our data facilitated the integration of the transcription map with the physical map around the DXS52 locus. Future analysis of the novel genes as candidates for Barth syndrome (BTHS) and chondrodysplasia punctata (CDPX2) is in progress.