ABSTRACT
BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach (AU)
Subject(s)
Animals , Mice , Dyskeratosis Congenita/chemically induced , Dyskeratosis Congenita/genetics , Dyskeratosis Congenita/metabolism , Dyskeratosis Congenita/diagnosisABSTRACT
We report on a gamma-ray coincidence analysis using a mixed array of hyperpure germanium and cerium-doped lanthanum tri-bromide (LaBr3:Ce) scintillation detectors to study nuclear electromagnetic transition rates in the pico-to-nanosecond time regime in 33,34P and 33S following fusion-evaporation reactions between an 18O beam and an isotopically enriched 18O implanted tantalum target. Energies from decay gamma-rays associated with the reaction residues were measured in event-by-event coincidence mode, with the measured time difference information between the pairs of gamma-rays in each event also recorded using the ultra-fast coincidence timing technique. The experiment used the good full-energy peak resolution of the LaBr3:Ce detectors coupled with their excellent timing responses in order to determine the excited state lifetime associated with the lowest lying, cross-shell, Iπ=4- "intruder" state previously reported in the N=19 isotone 34P. The extracted lifetime is consistent with a mainly single-particle M2 multipolarity associated with a f7/2âd5/2 single particle transition.
ABSTRACT
Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome associated with abnormalities of the skin, fingernails, and tongue. Other clinical manifestations may include epiphora, lung fibrosis, liver cirrhosis, osteoporosis, and a predisposition to develop a variety of malignancies. The clinical picture often resembles that of a premature aging syndrome and tissues affected are those with a high cell turnover. DC has been linked to mutations in at least four distinct genes, three of which have been identified. The product of these genes, dyskerin, the telomerase RNA (TERC), and the catalytic unit of telomerase (TERT) are part of a ribonucleoprotein complex, the telomerase enzyme, that is essential for the elongation and maintenance of chromosome ends or telomeres. All patients with DC have excessively short telomeres, indicating that the underlying defect in these individuals is an inability to maintain the telomeres. The purpose of the current review is to highlight recent insights into the molecular pathogenesis of DC. We discuss the impact these findings have on our current understanding of telomere function and maintenance, and on the diagnosis, management, and treatment of patients with conditions caused by dysfunctional telomeres.
Subject(s)
Chromosomes, Human, X/genetics , Dyskeratosis Congenita/genetics , Telomerase/genetics , Telomere/metabolism , Dyskeratosis Congenita/diagnosis , Dyskeratosis Congenita/therapy , Genetic Predisposition to Disease , Genetic Therapy , Humans , Mutation , Neoplasms/geneticsABSTRACT
We recorded the electroretinogram (ERG) from human subjects with normal vision, using ganzfeld stimulation in the presence of rod-suppressing blue background light. In families of responses to flashes of increasing intensity, we investigated features of both receptoral and post-receptoral origin. Firstly, we found that the oscillatory potentials (OPs, that have long been known to be post-receptoral) exhibited a time course that was invariant over a range of bright flash intensities. Secondly, we found that the photopic b-wave (which probably originates in cone ON bipolar cells) was most pronounced after test flashes of around 20 Td s, and could be suppressed either by increasing the test flash intensity or by applying a second flash after the test flash. We obtained estimates of the time course of the cone photoreceptor response using the paired-flash technique, in which an intense 'probe' flash was delivered at different times after a test flash. The response to the probe flash was recorded and, its amplitude was measured at early times after the probe flash. Estimates obtained in this way were of normalized amplitude, but could be scaled to an absolute amplitude by making an assumption about the level of probe-flash response that corresponded to complete suppression of photoreceptor current. For moderately bright test flashes the estimated cone photoreceptor response at early times coincided closely with the a-wave of the test flash ERG. However, the maximal size of this estimated response accounted for only about 70% of the peak a-wave amplitude in the case of bright flashes, and for an even smaller proportion after flashes of lower intensity, and we take this to indicate the existence of a third substantial post-receptoral contribution to the a-wave. For dim flashes, the time-to-peak of the cone response was around 15-20 ms, and for saturating flashes the dominant time constant of recovery was about 18 ms. The intensity dependence of the estimated cone response amplitude at fixed times followed an exponential saturation relation. We provide a comparison between our estimates of photoreceptor responses from human cones, and recent estimates from monkey cones obtained using related ERG approaches, and earlier single-cell measurements from isolated primate cones.
Subject(s)
Evoked Potentials, Visual/physiology , Retinal Cone Photoreceptor Cells/physiology , Vision, Ocular/physiology , Adult , Algorithms , Electrophysiology , Electroretinography/methods , Humans , Light , Photic StimulationABSTRACT
Dyskeratosis congenita is an inherited skin and bone marrow failure syndrome. There are X-linked, autosomal dominant and autosomal recessive forms of the disease. The X-linked form is due to mutations in the DKC1 gene at Xq28. The encoded protein, dyskerin, is a component of both small nucleolar ribonuclear protein particles and the telomerase complex. Mutations in DKC1 mainly lead to amino acid substitutions. The autosomal dominant form of the disease is due to mutations in hTR, the RNA component of telomerase, making it likely that the disease is due to defective telomerase activity. Mutations in hTR are predicted to either disrupt secondary structure or alter the template region. The gene or genes involved in the recessive forms of the disease remain elusive, though genes whose products are required for telomere maintenance are strong candidates.
Subject(s)
Dyskeratosis Congenita/genetics , Animals , Cell Cycle Proteins/genetics , Chromosome Mapping , Chromosomes, Human, X , Dyskeratosis Congenita/diagnosis , Dyskeratosis Congenita/physiopathology , Female , Genes, Dominant , Genes, Recessive , Humans , Male , Mice , Mutation , Nuclear Proteins/genetics , Pedigree , Telomerase/genetics , Telomerase/metabolismABSTRACT
Dyskeratosis congenita is a progressive bone-marrow failure syndrome that is characterized by abnormal skin pigmentation, leukoplakia and nail dystrophy. X-linked, autosomal recessive and autosomal dominant inheritance have been found in different pedigrees. The X-linked form of the disease is due to mutations in the gene DKC1 in band 2, sub-band 8 of the long arm of the X chromosome (ref. 3). The affected protein, dyskerin, is a nucleolar protein that is found associated with the H/ACA class of small nucleolar RNAs and is involved in pseudo-uridylation of specific residues of ribosomal RNA. Dyskerin is also associated with telomerase RNA (hTR), which contains a H/ACA consensus sequence. Here we map the gene responsible for dyskeratosis congenita in a large pedigree with autosomal dominant inheritance. Affected members of this family have an 821-base-pair deletion on chromosome 3q that removes the 3' 74 bases of hTR. Mutations in hTR were found in two other families with autosomal dominant dyskeratosis congenita.
Subject(s)
Chromosomes, Human, Pair 3 , Dyskeratosis Congenita/genetics , Mutation , RNA/genetics , Telomerase/genetics , Cell Line , Chromosome Mapping , DNA Mutational Analysis , Female , Genes, Dominant , Genetic Linkage , Humans , Male , Pedigree , Point Mutation , TelomereABSTRACT
BACKGROUND: Although asthma can be associated with significant airflow obstruction in those over the age of 65, it is often underdiagnosed and undertreated. OBJECTIVE: To describe severity of asthma, allergy skin test sensitivities, indoor allergen exposures, and the impact on quality of life (QOL) and health status in elderly persons with asthma. METHODS: A cross-sectional data analysis with 80 elderly persons with asthma recruited from medical, geriatric, and allergy/immunology tertiary care centers. Asthma severity was determined by symptoms and measurements of lung function. House dust specimens were collected from mattresses and bedroom carpets and analyzed separately for the major allergens of house dust, using monoclonal antibody-based immunoenzymetric assays. QOL was measured using Juniper's Asthma Quality of Life Questionnaire. Health status was measured using the Short Form Health Survey Medical Outcome Questionnaire which included Ferrans and Powers' Quality of Life Index subscales. RESULTS: Two-thirds of participants had either moderate or severe persistent asthma. Skin tests to a battery of common airborne allergens were positive to at least one allergen in 56 of the 75 participants tested (74.7%). Reservoir dust allergen levels were often high enough to place participants at risk of symptoms or at risk of developing sensitization. Increased asthma severity was associated with significantly lower QOL and a trend toward decreased health status. CONCLUSIONS: Asthma is a significant chronic problem in the elderly. Atopy was common. Asthma severity impacts on these participants' QOL and health status. Results support interventions aimed at identifying allergens precipitating attacks and reducing them in the home.
Subject(s)
Asthma/epidemiology , Hypersensitivity, Immediate/epidemiology , Aged , Air Pollution, Indoor/adverse effects , Air Pollution, Indoor/analysis , Allergens/adverse effects , Animals , Animals, Domestic , Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Asthma/etiology , Asthma/psychology , Baltimore/epidemiology , Bedding and Linens , Cats , Cockroaches/immunology , Dogs , Drug Utilization/statistics & numerical data , Dust , Environmental Exposure , Health Status Indicators , Housing , Humans , Humidity , Hypersensitivity, Immediate/etiology , Hypersensitivity, Immediate/psychology , Insect Proteins/adverse effects , Insect Proteins/immunology , Quality of Life , Severity of Illness Index , Skin Tests , Smoking/epidemiology , Spirometry , Urban PopulationABSTRACT
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.
Subject(s)
Cell Cycle Proteins/genetics , Dyskeratosis Congenita/genetics , Mutation , Nuclear Proteins/genetics , Base Sequence , Dyskeratosis Congenita/diagnosis , Dyskeratosis Congenita/physiopathology , Gene Expression , Genetic Testing , Humans , Male , Molecular Sequence Data , Polymorphism, Genetic , RNA, MessengerABSTRACT
Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which patients undergo premature ageing and have a predisposition to malignancy. X-linked and autosomal (dominant and recessive) forms of the disease are recognized. The gene responsible for X-linked DC (DKC1) encodes a highly conserved protein called dyskerin that is believed to be essential in ribosome biogenesis and may also be involved in telomerase RNP assembly. Here we show that in X-linked DC, peripheral blood cells have dramatically reduced telomere lengths but normal levels of telomerase activity. We also find that subjects with autosomal DC have significantly shorter telomeres than age-matched normal controls suggesting that both forms of the disease are associated with rapid telomere shortening in hemopoietic stem cells. The further characterization of these genes will not only lead to a better understanding of the biology of DC but may also provide further insights into the maintenance of telomeres and the biology of aplastic anemia, ageing, and cancer.
Subject(s)
Dyskeratosis Congenita/genetics , Telomere/genetics , Adolescent , Adult , Aging/genetics , Cell Cycle Proteins/genetics , Child , Child, Preschool , Dyskeratosis Congenita/blood , Dyskeratosis Congenita/pathology , Female , Humans , Male , Nuclear Proteins/genetics , Telomere/ultrastructureABSTRACT
Plasmodium falciparum glucose 6-phosphate dehydrogenase (Pf Glc6PD), compared to other Glc6PDs has an additional 300 amino acids at the N-terminus. They are not related to Glc6PD but are similar to a family of proteins (devb) of unknown function, some of which are encoded next to Glc6PD in certain bacteria. The human devb homologue has recently been shown to have 6-phosphogluconolactonase (6PGL) activity. This suggests Pf Glc6PD may be a bifunctional enzyme, the evolution of which has involved the fusion of adjacent genes. Further functional analysis of Pf Glc6PD has been hampered because parts of the gene could not be cloned. We have isolated and sequenced the corresponding Plasmodium berghei gene and shown it encodes an enzyme (Pb Glc6PD) with the same structure as the P. falciparum enzyme. Pb Glc6PD is 950 amino acids long with significant sequence similarity in both the devb and Glc6PD domains with the P. falciparum enzyme. The P. berghei enzyme does not have an asparagine-rich segment between the N and C halves and it contains an insertion at the same point in the Glc6PD region as the P. falciparum enzyme but the insertion in the P. berghei is longer (110 versus 62 amino acids) and unrelated in sequence to the P. falciparum insertion. Though expression of this enzyme in bacteria produced largely insoluble protein, conditions were found where the full-length enzyme was produced in a soluble form which was purified via a histidine tag. We show that this enzyme has both Glc6PD and 6PGL activities. Thus the first two steps of the pentose phosphate pathway are catalysed by a single novel bifunctional enzyme in these parasites.
Subject(s)
Carboxylic Ester Hydrolases/metabolism , Glucosephosphate Dehydrogenase/metabolism , Phosphogluconate Dehydrogenase/metabolism , Plasmodium berghei/enzymology , Plasmodium falciparum/enzymology , Amino Acid Sequence , Animals , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Molecular Sequence DataABSTRACT
Glucose-6-phosphate dehydrogenase (G6PD) is expressed in all tissues, where it catalyses the first step in the pentose phosphate pathway. G6PD deficiency is prevalent throughout tropical and subtropical regions of the world because of the protection it affords during malaria infection. Although most affected individuals are asymptomatic, there is a risk of neonatal jaundice and acute haemolytic anaemia, triggered by infection and the ingestion of certain drugs and broad beans (favism). A rare but more severe form of G6PD deficiency is found throughout the world and is associated with chronic non-spherocytic haemolytic anaemia. Many deficient variants of G6PD have been described. DNA sequence analysis has shown that the vast majority of these are caused by single amino acid substitutions. The three-dimensional structure of G6PD shows a classical dinucleotide binding domain and a novel beta + alpha domain involved in dimerization.
Subject(s)
Glucosephosphate Dehydrogenase Deficiency , Anemia, Hemolytic, Congenital Nonspherocytic/enzymology , Anemia, Hemolytic, Congenital Nonspherocytic/prevention & control , Anemia, Hemolytic, Congenital Nonspherocytic/therapy , Glucosephosphate Dehydrogenase/chemistry , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase/physiology , Glucosephosphate Dehydrogenase Deficiency/diagnosis , Glucosephosphate Dehydrogenase Deficiency/genetics , Humans , Infant, Newborn , Jaundice, Neonatal/enzymology , Malaria/enzymology , Malaria/prevention & control , Models, MolecularABSTRACT
Serotonin syndrome is an underreported complication of pharmacotherapy that has been relatively ignored in the medical literature. We discuss 2 recent cases seen at our institution and 39 cases described in the English-language literature since 1995. We found that patients with serotonin syndrome most often (74.3%) presented within 24 hours of medication initiation, overdose, or change in dosage. The most common presenting symptoms and signs were confusion, agitation, diaphoresis, tachycardia, myoclonus, and hyperreflexia. The prevalences of hypertension, coma/unresponsiveness, seizures, and death were not as prominent in our study as previously reported, perhaps reflecting earlier recognition and intervention. The most common therapeutic intervention was supportive care alone (48% of patients). The use of 5-hydroxytryptamine (5-HT) antagonists such as cyproheptadine, however, has become more common and might reduce the duration of symptoms. Only 1 death occurred, and most patients (57.5%) had complete resolution of their symptoms within 24 hours of presentation. The increased use of serotonergic agents (alone and in combination) across multiple medical disciplines presents the possibility that the prevalence and clinical significance of this condition will rise in the future. Internists will need to be increasingly aware of and prepared for this pharmacologic complication. Prevention, early recognition of the clinical presentation, identification and removal of the offending agents, supportive care, and specific pharmacologic therapy are all important to the successful management of serotonin syndrome.
Subject(s)
Serotonin Syndrome/physiopathology , Adolescent , Adult , Aged , Aged, 80 and over , Antidepressive Agents/adverse effects , Child , Child, Preschool , Cognition Disorders/etiology , Depressive Disorder/drug therapy , Diagnosis, Differential , Female , Humans , Male , Middle Aged , Reflex, Abnormal , Serotonin Syndrome/etiologyABSTRACT
In this study, we present evidence that PI 3-kinase is required for alpha-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) pathway controls transit through G(1) phase of the cell cycle by regulating the induction of cyclin D1 mRNA levels and cyclin dependent kinase 4 (CDK4)-cyclin D1 activity. In IIC9 cells, PI 3-kinase activation also is an important controller of the expression of cyclin D1 protein and CDK4-cyclin D1 activity. Pretreatment of IIC9 cells with the selective PI 3-kinase inhibitor, LY294002 blocks the alpha-thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect alpha-thrombin-induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant-negative Ras significantly decreased both alpha-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the alpha-thrombin-induced Ras activation coordinately regulates ERK and PI 3-kinase activities, both of which are required for expression of cyclin D1 protein and progression through G(1).
Subject(s)
Cyclin D1/biosynthesis , Cyclin-Dependent Kinases/metabolism , G1 Phase , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins , Thrombin/metabolism , Animals , Cells, Cultured , Chromones/pharmacology , Cricetinae , Cricetulus , Cyclin-Dependent Kinase 4 , DNA Replication , Enzyme Inhibitors/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Morpholines/pharmacology , Phosphoinositide-3 Kinase InhibitorsABSTRACT
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.
Subject(s)
Anemia, Aplastic/genetics , Cell Cycle Proteins/genetics , Cerebellum/abnormalities , Immune System Diseases/genetics , Mutation, Missense/genetics , Nuclear Proteins/genetics , Amino Acid Substitution/genetics , Female , Fetal Growth Retardation/genetics , Humans , Infant , Male , Microcephaly/genetics , Pedigree , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , SyndromeABSTRACT
We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.
Subject(s)
Anemia, Hemolytic/genetics , Glucosephosphate Dehydrogenase/genetics , Granulocytes/physiology , Adult , Anemia, Hemolytic/complications , Anemia, Hemolytic/enzymology , Anemia, Hemolytic/physiopathology , Chronic Disease , Communicable Diseases/etiology , Communicable Diseases/genetics , Enzyme Activation , Female , Genetic Predisposition to Disease , Glucosephosphate Dehydrogenase/metabolism , Humans , Mutation , Pedigree , Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Cell Cycle Proteins/genetics , Dyskeratosis Congenita/genetics , Germ-Line Mutation , Nuclear Proteins/genetics , Sequence Deletion , X Chromosome , 3' Untranslated Regions/genetics , Amino Acid Sequence , Base Sequence , Female , Haplotypes , Humans , Male , Molecular Sequence Data , PedigreeABSTRACT
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.
Subject(s)
Cell Cycle Proteins/genetics , Dyskeratosis Congenita/genetics , Hydro-Lyases , Mutation, Missense , Nuclear Proteins/genetics , Ribonucleoproteins, Small Nuclear , Saccharomyces cerevisiae Proteins , X Chromosome , Amino Acid Sequence , Cell Cycle Proteins/chemistry , Female , Humans , Male , Microtubule-Associated Proteins/chemistry , Models, Genetic , Molecular Sequence Data , Nuclear Proteins/chemistry , Pedigree , Polymorphism, Genetic , Polymorphism, Single-Stranded Conformational , RNA-Binding Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Using the published protein sequence from a rabbit microsomal glucose-6-phosphate dehydrogenase G6PD we have isolated and sequenced a cDNA clone coding for its human equivalent, which is also known as hexose-6-phosphate dehydrogenase (H6PD) and glucose dehydrogenase. The corresponding genomic sequence is in the databases enabling its localization to chromosome 1p36. The gene spans 37 kb and consists of 5 exons, the fifth of which codes for more than half of the 89 kDa protein. The first intron is a 10 kb insertion in the 5' untranslated sequence. The predicted mRNA has an exceptionally long (6.5 kb) 3' untranslated sequence. The predicted protein shows extensive homology with X-linked G6PD, suggesting the two genes share a common ancestor but no intron positions are conserved between the two genes suggesting the gene duplication was an ancient event. The C-terminal portion of the protein is not homologous with G6PD but shows limited homology with proteins of unknown function found throughout evolution and encoded next to G6PD in various micro-organisms. Intriguingly this C-terminal portion has some homology with the N-terminal sequence of Plasmodium falciparum G6PD.