Biomedical subjects
Publications and source records attributed to G Loudianos.
Wilson's disease (WD) is an autosomal recessive disorder of copper transport, related to mutations of the ATP7B gene (McKusick 277900). Here we report a new case of WD in which a rare mutation, Leu492Ser expressed for the first time in homozygosity, is associated with neurological presentation of the disease and arylsulfatase A pseudodeficiency.
More than 200 Wilson disease (WD) disease-causing mutations have been defined to date. Missense mutations are largely prevalent while splice-site mutations are limited in number. Most reside in the splice donor or acceptor sites and only a minority are detected in splicing consensus sequences. Furthermore, only a few splicing mutations have been studied at the RNA level to date. In this study, using the RT-PCR method we performed the molecular characterization of four consensus splice-site mutations identified by DNA analysis in patients with WD. One of them, previously described 1707+3insT, occurred at position 3 in the donor splice site of intron 4, while the other three, 2122-8T>G, 2866-6T>G, and 3061-12T>A, are novel and occurred in the acceptor splice sites of introns 7, 12, and 13, respectively. Analysis revealed a prevalently abnormal splicing in the samples carrying the mutations compared to the normal controls. Comparison of RNA splicing with normal controls in liver and lymphocytes further suggests that abnormal splicing of the WD gene is also present and differentially regulated in normal tissues. The results produced in this study strongly suggest that DNA mutations residing in the consensus sequence of WD gene splice sites result in the WD phenotype by interfering with the production of the normal WD protein. Further studies are necessary to better quantify the amount of different transcripts produced by these mutations, and establish their correlation with the disease phenotype.
Wilson's disease is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase encoded on chromosome 13. This ATPase is expressed in hepatocytes where it is localized to the trans-Golgi network and transports copper into the secretory pathway for incorporation into ceruloplasmin and excretion into the bile. Under physiologic circumstances, biliary excretion represents the sole mechanism for copper excretion, and thus affected individuals have progressive copper accumulation in the liver. When the capacity for hepatic storage is exceeded, cell death ensues with copper release into the plasma, hemolysis, and tissue deposition. Presentation in childhood may include chronic hepatitis, asymptomatic cirrhosis, or acute liver failure. In young adults, neuropsychiatric symptoms predominate and include dystonia, tremor, personality changes, and cognitive impairments secondary to copper accumulation in the central nervous system. The laboratory diagnosis of Wilson's disease is confirmed by decreased serum ceruloplasmin, increased urinary copper content, and elevated hepatic copper concentration. Molecular genetic analysis is complex as more than 100 unique mutations have been identified and most individuals are compound heterozygotes. Copper chelation with penicillamine is an effective therapy in most patients and hepatic transplantation is curative in individuals presenting with irreversible liver failure. Elucidation of the molecular genetic basis of Wilson's disease has permitted new insights into the mechanisms of cellular copper homeostasis.
In this study, we report the further results of an ongoing project on the delineation of the spectrum of mutations on the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analyzed 24 additional families and detected 16 mutations (five frameshifts, two splice site, two nonsense, and seven missense), of which six are novel. On adding these results to the ones already published by us, we conclude that WD shows a marked allelic heterogeneity in the Greek population. Of the total number of mutations so far detected, the most common eight account for the molecular defect in 72.8% of the WD chromosomes. The most frequent mutation is the His0169Gln, which has a frequency of 28.5%, similar to those reported in North European populations. Using these data, an efficient strategy of mutation screening for WD is possible in this population, thus improving the possibility of preclinical diagnosis.
We have studied a patient with Wilson disease (WD), belonging to a family segregating late-onset, dominant cerebellar ataxia. Analysis of the WD gene showed that the patient is a compound heterozygote, carrying the 14His1069Gln mutation from the father and the 8Gly710Ser mutation from the mother. The 8Gly710Ser is a mutation described previously only in a Swedish patient. Our patient is also homozygous for arylsulfatase A pseudodeficiency. This genetic defect, which has been reported in association with other neuropsychiatric syndromes, has not been described in WD.
Wilson disease (WD) in the Sardinian population has an approximate incidence of 1:7,000 live births. Mutation analysis of the WD gene in this population reported in our previous articles led us to the characterization of two common mutations and a group of 13 rare mutations accounting for the molecular defect of 8.5, 7.9, and 15.1% of the WD chromosomes. However, molecular analysis of the WD chromosomes containing the most common haplotype, which accounts for 60.5% of the WD chromosomes, failed to define the disease-causing mutation. In this study, we characterized the promoter and the 5' UTR of the WD gene sequence and carried out a mutation analysis in this DNA region from patients with the most common haplotype. The promoter is contained in a GC-rich island and shows a TATA and a CAAT consensus sequence as well as potential binding sites for transcription factors and metal response elements. In all the analyzed 92 chromosomes with this haplotype, we detected a single mutation consisting of a 15-nt deletion from position -441 to position -427 relative to the translation start site. Expression assays demonstrated a 75% reduction in the transcriptional activity of the mutated sequence compared to the normal control. By adding this mutation to those that have been already characterized, we have now defined the molecular defect in 92% of the WD chromosomes in Sardinians. The high frequency, the expected prevention by preclinical diagnosis and early treatment of the devastating effect of WD on the nervous system and liver tissue, and the feasibility to detect most of molecular defects by DNA analysis indicate that WD in the Sardinian population should be added to the list of diseases currently detected by newborn screening.
In this study, we report further results of mutation analysis of the ATP7B gene in Wilson disease (WD) patients of Mediterranean origin. A total of 136 WD chromosomes, 73 of which were of Italian, 43 of Turkish, 18 of Sardinian, and two of Spanish origin, were analysed and the mutation characterised in 84.5% of them. We found 50 different mutations of which 19 are novel, including three nonsense, one frameshift, and 15 missense mutations. The mutations detected were rare and mostly found in the compound heterozygous state together with other mutations and only rarely in homozygosity. Most of these mutations lie in the transmembrane and ATP binding loop regions. These data expand our knowledge of both the structure-function relationships of the WD protein and the molecular pathology of WD, thus improving our capability of prevention and genetic counselling.
This study presents the update results of an ongoing project on the delineation of the spectrum of mutations at the Wilson disease (WD) gene in WD patients of Mediterranean origin. In studying 59 patients, of whom were 26 Continental Italians, 22 Sardinians, 9 Turkish, and 2 Albanians, we have found 31 novel and three known mutations. Of the novel mutations, 3 are deletions, two nonsense, 2 splice or consensus splice site, and 24 missense. The large majority of the missense mutations lie in evolutionary conserved regions of the WD gene of documented functional importance. Most of our patients were compound heterozygotes, and only a few were homozygotes. In addition, three polymorphisms were detected. By adding the new data to those previously reported by our group, we have to date detected 85% of mutations in the WD chromosomes from Continental Italians, 30% from Sardinians, 81.7% from Turkish and 66.7% from Albanians. Most of the mutations characterized are rare, and only a limited number are common. Of the common mutations 5 were found in Continental Italians, two in Sardinians and a single one in Turkish. Because there are so many causative mutations of the disease, the preclinical and prenatal diagnosis of WD should be carried out by a combination of mutation and linkage analysis.
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BACKGROUND/AIMS: An improved method for the study of copper metabolism in Wilson's disease, using a stable, rather than radioactive, copper isotope (65Cu) has recently been described. We report on the use of this method for the study of a family with two members affected by Wilson's disease. SUBJECTS: The family comprised parents and four siblings: one 20-year-old male and three females, aged 22, 17 and 5 years, respectively. The boy and the 17-year-old girl both presented with liver cirrhosis. Diagnosis of Wilson's disease was suggested by elevated liver copper content and/or low caeruloplasmin levels and Kayser-Fleischer ring. METHODS: All family members were given an oral dose of 3 mg of 65Cu. Blood samples were taken at 0, 1, 2, 6, 24, 48, and 72 hours. In 4 subjects, additional blood samples were drawn at 7, 14 and 21 days after dosage. The ratio 65Cu:63Cu in serum was determined in all samples by means of Inductively Coupled Plasma Mass Spectrometry. RESULTS: The diagnosis of Wilson's disease was confirmed in the two symptomatic members by the unequivocal decrease observed in the 65Cu percent enrichment, which approached zero by 72 hours. In contrast, Wilson's disease could be definitely excluded in both siblings, one of whom only 5 years old, on the evidence of net secondary peaks, showing normal incorporation of 65Cu into caeruloplasmin. These findings were later confirmed by genetic analysis. Parents, who carried defective genes with different mutations, also showed different abnormalities of copper metabolism. CONCLUSIONS: The oral test with the stable copper isotope 65Cu is a safe, non invasive option able to exclude Wilson's disease in patients with a difficult diagnosis or in a presymptomatic stage. However, positive tests must still be confirmed by copper dosage in liver biopsies, as heterozygotes can present with severe alterations of copper metabolism, without developing symptoms of the disease. The use of this test in conjunction with genetic analysis on a larger number of heterozygous subjects may add to the understanding of the Wilson's disease defect.
This study reports 12 novel mutations of the Wilson disease (WD) gene which have been detected by the molecular analysis of 29 patients of Mediterranean descent carrying uncommon chromosomal haplotypes at the WD locus. These mutations include two nonsense, one splice site and nine missense. The missense mutations lie in regions of the WD gene critical for its function, such as the transmembrane region, the transduction domain and the ATP loop and ATP-binding domain, indicating that they are disease-causing mutations. These new findings improve our knowledge for the role played by functional domains on the ATP7B function.
We analyzed mutations and defined the chromosomal haplotype in 127 patients and Mediterranean descent who were affected by Wilson disease (WD), 39 Sardinians, 49 Italians, 33 Turks, and 6 Albanians. Haplotypes were derived by use of the microsatellite markers D13S301, D13S296, D13S297, and D13S298, which are linked to the WD locus. There were five common haplotypes in Sardinians, three in Italians, and two in Turks, which accounted for 85%, 32%, and 30% of the WD chromosomes, respectively. We identified 16 novel mutations: 8 frameshifts, 7 missense mutations, and 1 splicing defect. In addition, we detected the previously described mutations: 2302insC, 3404delC, Arg1320ter, Gly944-Ser, and His1070Gin. Of the new mutations detected. two, the 1515insT on haplotype I and 2464delC on haplotype XVI, accounted for 6% and 13%, respectively, of the mutations in WD chromosomes in the Sardinian population. Mutations H1070Q, 2302insC, and 2533delA represented 13%, 8%, and 8%, respectively, of the mutations in WD chromosomes in other Mediterranean populations. The remaining mutations were rare and limited to one or two patients from different populations. Thus, WD results from some frequent mutations and many rare defects.
The term delta beta-thalassemia with normal HbF has been recently proposed to define heterogenous delta and beta globin gene molecular defects involving the same chromosome in cis. Here, we describe a Sardinian family in which three members showing microcytosis, border-line HbA2 levels and normal HbF proved to be heterozygotes for delta(+) 27 and beta(0) 39 point mutations in cis by allele specific oligonucleotide hybridization as well as by ECO 0 109 I endonuclease digestion and electrophoresis. As some of these beta-thalassemia carriers shows normal HbA2 levels, knowledge of the molecular basis of this novel delta beta-thalassemia silent phenotype would be useful in thalassemia screening and genetic counselling.
This paper describes a case of prenatal diagnosis for Wilson disease (WD) carried out in an at-risk couple of Sardinian descent, following non-directive genetic counselling. Diagnosis was obtained by using eight microsatellites located within or flanking the WD locus, six of which were 100 per cent and two 50 per cent informative. The use of several markers may limit the occurrence of misdiagnosis resulting from recombination or instability of repeats.
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We have previously described a family of Northern Sardinian descent in which the propositus was affected by thalassemia major resulting from compound heterozygosity for codon 39 nonsense mutation and the beta +IVS II nt 745 mutation and in which all heterozygotes for the beta +IVS II nt 745 mutation had normal hemoglobin (Hb) A2 levels. To define the reasons for normal HbA2 levels in otherwise typical beta-thalassemia heterozygotes, we cloned and sequenced the delta-thalassemia gene in cis to the beta +IVS II nt 745 mutation. The sequence analysis showed a single nucleotide substitution (G----A) at position 69 nts (delta +69) downstream to the polyA addition site. Dot blot analysis with an oligonucleotide probe complementary to the delta +69 mutation detected this mutation in several heterozygotes for the beta +IVS II nt 745 mutation from the proband's family, but failed to show it either in a group of normal individuals of the same origin or in nonrelated heterozygotes for the beta +IVS II nt 745 mutation of the same or different descent from the proband. The delta +69 (G----A) mutation may be responsible for the low delta-globin output from the beta +IVS II nt 745 chromosome or could be a silent polymorphism not affecting the function of the delta-globin gene. The normal G at position 69 is part of a sequence very similar to the core DNA (A/T)GATA(A/G) motif (GATA box) that is a binding site for the GATA-1 protein. Gel-retardation assay has shown that a DNA fragment containing the GATA motif with the G----A at position +69 has increased binding affinity for erythroid-specific DNA binding protein(s) as compared with the wild-type sequence. These findings may suggest that the delta +69 mutation is responsible for the deficient function of the in cis delta-globin gene.
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This paper describes a family of Central Italian origin in which three patients in two generations had either thalassaemia intermedia or a late presenting form of thalassaemia major. Sequence analysis of the patients' DNA revealed that only one of the beta-globin genes was affected by a beta-thalassaemia mutation (the codon 39 nonsense mutation), the other being completely normal, apart from the complex rearrangement (-T +ATA) at position -530 5' to the CAP site of the beta-globin gene, which has uncertain clinical significance. Haematologically, all these patients were characterized by unusually low HbF levels (1.8-7.3%) for a beta-thalassaemia major or intermedia phenotype. The mother of the two patients with thalassaemia intermedia was heterozygous for beta-thalassaemia (codon 39 nonsense mutation), while the father had thalassaemia-like red cell indices, an increased alpha/non alpha chain synthesis ratio, a slight increase of HbF and a low HbA2 level, but showed entirely normal beta-globin gene sequences, apart from the complex rearrangement (-T +ATA) at position -530 5' to the CAP site. One of the thalassaemia intermedia patients married a normal woman and they had a child with thalassaemia major who inherited only the codon 39 nonsense mutation but not the complex rearrangement at position -530. The clinical phenotype of thalassaemia-intermedia or major in the patients from this family may be explained by postulating the inheritance of the double heterozygous state for beta-thalassaemia and for a mutation in a gene coding for an erythroid-specific DNA binding protein which may impair the function of the normal beta-globin gene. Heterozygosity for this postulated mutation (father of the patients with thalassaemia intermedia) may result in the production of a beta-thalassaemia carrier state with normal HbA2 level.