Georgios Loudianos

Molecular characterization of Wilson disease in the Sardinian population—Evidence of a founder effect

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. Hum Mutat 14:294–303, 1999.

Mutation analysis in patients of Mediterranean descent with Wilson disease: identification of 19 novel mutations.

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.

Further delineation of the molecular pathology of Wilson disease in the Mediterranean population

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. Hum Mutat 12:89–94, 1998.

The C–T substitution in the distal CACCC box of the β-globin gene promoter is a common cause of silent β thalassaemia in the Italian population

This paper describes four families of Italian descent in each of which the propositus had the clinical phenotype of thalassaemia intermedia, resulting from the compound heterozygous state for high HbA2β thalassaemia and type I silent β thalassaemia. Direct sequencing on amplified DNA and/or oligonucleotide analysis detected, in all families but one, the compound heterozygous state for codon 39 nonsense mutation and the C‐T substitution at position – 101 in the distal CACCC box of the β‐globin gene promoter (βth–101). Members of these families who are heterozygous for high HbA2β thalassaemia showed the codon 39 nonsense mutation, while those with the clinical phenotype of silent β thalassaemia had the βth–101 mutation. In the remaining family, the propositus and one of his siblings had the compound heterozygous state for a molecularly undefined high HbA2β thalassaemia and the βth–101 mutation in combination with the triple α globin gene arrangement. These patients showed a more severe thalassaemia intermedia like clinical phenotype as compared to those with the same β‐globin genotype and a normal α‐globin gene arrangement. In the families investigated the βth–101 was always associated with haplotype I. A group of patients with thalassaemia intermedia from Southern Italy, either homozygous or heterozygous for haplotype I and in whom previous studies had failed to define the mutation in one of the β thalassaemia globin genes, were screened by oligonucleotide analysis for the presence of the βth–101. Three out of nine were positive. These results indicate that the βth–101 mutation is a common cause of the type I silent β thalassaemia phenotype in the Southern Italian population.

Characterization of the Molecular Defect in the ATP7B Gene in Wilson Disease Patients from Yugoslavia

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase (ATP7B). Approximately 150 mutations of the ATP7B have been identified to date. In this paper, we report the results of molecular characterization and genotype-phenotype analysis, which we have carried out on 35 patients from Yugoslavia affected by WD. Using single-strand conformational polymorphism (SSCP) followed by direct sequencing, we characterized the molecular defect in 80% of WD chromosomes and found 11 different mutations, three of which are novel. The most common mutations that accounted for the molecular defect in 71.3% of WD chromosomes were H1069Q (48.9%), 2304-2305insC (11.4%), R616Q (5.7%), and A1003T (5.7%). The results produced in this paper indicate that the best strategy for mutation detection in Yugoslavian patients with WD is an SSCP analysis of exons 14, 8, 5, and 13, where most of the defects (73.1%) lie, followed by mutation analysis of the remaining exons in ATP7B in patients in whom the mutation was not detected by the finitial screening. These data can be used to develop straightforward genetic testing in this population or in other countries composed of a genetically mixed population like the United States, where a significant number of immigrants came from Central and Eastern Europe.

A β‐thalassaemia phenotype not linked to the β‐globin cluster in an Italian family

Summary. 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’ UNA revealed that only one of the β‐globin genes was affected by a β‐thalassaemia mutation (the codon 39 nonsense mutation), the other being completely normal, apart from the complex rearrangement (‐T + ATA) at position −5305’ to the CAP site of the β‐globin gene, which has uncertain clinical significance. Haematologically, all these patients were characterized by unusually low HbF levels (1.8–7.3%) for a β‐thalassaemia major or intermedia phenotype. The mother of the two patients with thalassaemia intermedia was heterozygous for β‐thalassaemia (codon 39 nonsense mutation), while the father had thalassaemia‐like red cell indices, an increased α/nonα chain synthesis ratio, a slight increase of HbF and a low HbA2 level, but showed entirely normal β‐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 β‐thalassaemia and for a mutation in a gene coding for an erythroid‐specific DNA binding protein which may impair the function of the normal β‐globin gene. Heterozygosity for this postulated mutation (father of the patients with thalassaemia intermedia) may result in the production of a β‐thalassaemia carrier state with normal HbA2 level.

Bannayan-Riley-Ruvalcaba syndrome with reactive nodular lymphoid hyperplasia and autism and a PTEN mutation.

Loredana Boccone,* Valentina Dessı̀, Antonietta Zappu, Silvia Piga, Maria Bonaria Piludu, Marco Rais, Carlo Massidda, Stefano De Virgiliis, Antonio Cao, and Georgios Loudianos Ospedale Regionale Microcitemie, Cagliari, Italy Dipartimento di Scienze Biomediche e Biotecnologie, USC, Italy Istituto di Neurogenetica e Neurofarmacologia, CNR-Cagliari, Italy U.O. Anatomia Patologica, Ospedale Businco, Cagliari, Italy Chirurgia Oncologica, Ospedale Businco, Cagliari, Italy

Allan–Herndon–Dudley syndrome (AHDS) caused by a novel SLC16A2 gene mutation showing severe neurologic features and unexpectedly low TRH-stimulated serum TSH

Thyroid hormones are known to be essential for growth, development and metabolism. Recently mutations in the SLC16A2 gene coding for the monocarboxylate thyroid hormone transporter 8, MCT8, have been associated with Allan-Herndon-Dudley syndrome (AHDS), an X-linked condition characterized by severe mental retardation, dysarthria, athetoid movements, muscle hypoplasia and spastic paraplegia. Here we describe in detail the clinical and biochemical features in a boy affected by AHDS with severe neurological abnormalities and a novel de novo SLC16A2 gene insertion, 1343-1344insGCCC, resulting in a truncated protein lacking the last four transmembrane domains (TMDs) as well as the carboxyl cytoplasmic end. He presents mental retardation, axial hypotonia, hypertonia of arms and legs, paroxysmal dyskinesias, seizures. The endocrine phenotype showed low serum total and free thyroxine (T4), very elevated total and free triiodothyronine (T3) and normal thyrotropin (TSH) with blunted response to thyrotropin-releasing hormone (TRH). The latter finding was unexpected and suggested that the lack of functional MCT8 was counterbalanced at the thyrotrope cell level by high serum T3 concentration and/or by increased intrapituitary type 2 deiodinase (D2) activity. Our case constitutes a relevant contribution to better characterize this disorder and to elucidate the functional consequences of SLC16A2 gene mutations.

Delineation of the spectrum of Wilson disease mutations in the Greek population and the identification of six novel mutations.

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.

Efficient strategy for molecular diagnosis of wilson disease in the Sardinian population

Wilson disease (WD) is an autosomal recessive disorder of copper transport resulting from the defective function of a copper-transporting ATPase (ATP7B) (1)(2)(3). More than 200 disease-causing mutations have been identified (4). In the Sardinian population, WD has an incidence of ∼1 in 7000 live births (5). Using single-strand conformation polymorphism (SSCP) and sequencing methods for mutation analysis, we have characterized 92% of the chromosomes analyzed and identified 16 different WD-causing mutations, 6 of which (−441/−427del, 213–214delAT, 1512–1513insT, R778W, 2463delC, and V1146M) are relatively common and account for 85% of chromosomes (6). On the basis of these data, we developed a reverse dot-blot (RDB) method as a practical solution to mutation screening in this population. DNA samples from Sardinian WD patients carrying different combinations of the most common mutations (−441/−427del, 2463delC, V1146M, 213–214delAT, 1512–1513insT, and R778W) were used as controls. Our aim was to obtain the same PCR conditions for all six pairs of primers that were used to amplify the regions containing the six most common mutations. We therefore designed primers with an identical melting temperatures and tested their specificity first in single and then in multiplex PCRs. We also wanted to obtain relatively equal yields for all PCR products to obtain comparable signals using the RDB method. We therefore tested different concentrations for each pair of primers and finally established primer concentrations that allowed us to obtain approximately …