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R Galanello

Publications and source records attributed to R Galanello.

At least 37 records · Page 2Linked to original sources
PubMed1998

Heterozygous beta-thalassemia with thalassemia intermedia phenotype.

In this study we investigated the molecular bases of the beta-thalassemia intermedia phenotype in six patients belonging to two unrelated families of Sardinian descent. Sequence analysis of the beta globin gene from these patients detected, as the sole abnormality, the heterozygosity for the codon 39 nonsense mutation. The A gamma and Ggamma promoters as well as the HS2 and HS3 core sequences of the beta globin LCR from these patients, did not show any non-polymorphic nucleotide variation from the consensus sequence. One of the parents was heterozygous for codon 39 nonsense mutation but showed the beta-thalassemia carrier phenotype; the other was hematologically normal and had an entirely normal beta globin gene sequence. In both families, other members showed the typical hematological phenotype, clinically silent, of heterozygous beta thalassemia. To explain the thalassemia intermedia phenotype, we postulated the presence of an unknown molecular defect interacting with the beta globin gene mutation. Haplotype analysis excluded that this postulated defect lies in the beta globin gene cluster.

Adolescent↗
PubMed1998

Evaluation of the BeTha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations.

We describe the evaluation of the Bio-Rad BeTha Gene 1 kit (Bio-Rad Laboratories, Hercules, CA), a DNA-probe assay designed for the qualitative determination of the eight most common Mediterranean beta-thalassemia mutations. The kit utilizes the principle of allele-specific oligonucleotide (ASO) hybridization. Following sample preparation and in vitro DNA amplification by the polymerase chain reaction (PCR), an allele-specific detection of the amplified products by a nonradioactive enzymatic assay is performed. Genomic DNA is prepared from an individual's whole blood with a DNA purification matrix. In a second step, the beta-globin gene is amplified in a multiplex PCR reaction containing four 5' biotinylated oligonucleotide primers. In a final step, an aliquot of the PCR reaction is first chemically denatured and then captured in two eight-well strips of a 96-well enzyme-linked immunosorbent assay (ELISA) plate by hybridization to an immobilized ASO probe. Each DNA sequence at each of the eight mutation sites is represented by one normal and one mutant ASO. During this capture/hybridization step, which is performed at 37 degrees C, only perfectly matched PCR products will be captured by an ASO. Subsequently, the allele-specific captured biotin-labeled PCR products are detected by a colorimetric enzymatic reaction. The system permits the detection of 16 beta-thalassemia alleles using a high-throughput format that can be automated easily. A clinical feasibility study was performed to evaluate the functionality (method comparison study, assay validity using samples previously collected and stored at various temperatures for different periods of time, interference on kit performance, and assay validity for prenatal diagnosis) and the usability (ease of use, sample throughput) of the kit. The analysis of 110 samples previously studied with reference methods showed 100% clinical sensitivity and specificity. We demonstrate here that the procedure not only increases the throughput of beta-thalassemia allele genotyping but also provides an accurate, rapid, reliable, and nonisotopic diagnostic tool.

Alleles↗
PubMed1998

Alpha-thalassemia carrier identification by DNA analysis in the screening for thalassemia.

Differentiation between heterozygous alpha-thalassemia and several phenotypically resembling alleles at the beta-globin gene cluster such as coinherited delta- and beta-thalassemia or gammadelta beta-thalassemia is a critical step in genetic counseling. In this paper we report our experience in the identification of the alpha-thalassemia carrier state using polymerase chain reaction (PCR)-based methods, and the feasibility and simplification of screening for thalassemia using this approach. Alpha-globin genotype was determined by PCR-based method in 526 adult subjects with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal hemoglobin A2 and F, and normal serum iron. To verify the reliability of the protocol used, in 68 of these subjects we performed globin chain synthesis analysis and in 101 we determined alpha-globin genotype by Southern blot analysis. Five hundred twenty-one (99%) of 526 subjects examined were identified as carriers of one or two alpha-thalassemia alleles. The identification of the alpha-thalassemia carrier state may be fast and accurate by PCR-based method, avoiding other cumbersome and expensive methods such as globin chain synthesis and Southern blot analysis.

Adult↗
PubMed1998

Identification of a new SCN5A mutation, D1840G, associated with the long QT syndrome. Mutations in brief no. 153. Online.

The long QT syndrome (LQT) is an inherited cardiac disorder that can cause sudden cardiac death among apparently healthy young individuals due to malignant ventricular arrhythmias. LQT was found to be caused by mutations in four genes LTQ1, LQT2, LQT3 and LQT5, and linkage was reported for an additional locus, LQT4, on chromosome 4q25-27. We have studied a large (n=131) LQT-affected Jewish kindred and identified tight linkage between the LQT-affected status and LQT3 (lod score 6.13, with an estimated recombination fraction of zero). We identified a new point-mutation, A to G substitution at nucleotide 5519 of the SCN5A gene, changing the aspartate 1840 to glycine, D1840G. This is a non-conservative change of an amino acid completely conserved in sodium channels from Molusca to human. The mutation was identified in all affected individuals (n=23), and not identified in all the unaffected family members (n=40), and not in 200 chromosomes of healthy control individuals. The mutation was identified in 3/12 individuals with equivocal phenotype, thus, providing an accurate dignostic tool for all family members. This mutation is currently being used in a cellular electrophysiological model, to characterize the function of the mutated sodium channel in this syndrome.

Amino Acid Substitution↗
PubMed1998

Prenatal diagnosis and screening of the haemoglobinopathies.

This paper reviews the most important aspects of carrier detection procedures, genetic counselling, population screening and prenatal diagnosis of the thalassaemias and sickle cell anaemia. Carrier detection can be made retrospectively, following the birth of an affected child, or prospectively. Carrier detection and genetic counselling in at-risk populations for alpha-thalassaemia and sickle cell anaemia is carried out mostly retrospectively. However prospective carrier screening is ongoing in Cuba and Guadeloupe for sickle cell anaemia and, in a very limited way, in some South East Asian populations, for alpha-thalassaemia. For beta-thalassaemia, several programmes, based on carrier screening and counselling of couples at marriage, preconception or early pregnancy, are operating in several Mediterranean at-risk populations. These programmes have been very effective, as indicated by increasing knowledge on thalassaemia and its prevention by the target population and by the marked decline of the incidence of thalassaemia major. Carrier detection is carried out by haematological methods followed by mutation detection by DNA analysis. Prenatal diagnosis is accomplished by mutation analysis on PCR-amplified DNA from chorionic villi. Future prospects include automation of the process of mutation-detection, simplification of preconception and preimplantation diagnosis and fetal diagnosis by analysis of fetal cells in maternal circulation.

Anemia, Sickle Cell↗
PubMed1998

Elevation of serum creatine kinase as the only manifestation of an intragenic deletion of the dystrophin gene in three unrelated families.

This study reports three children from three unrelated families, aged from 9 to 12 years, who were investigated because of the incidental finding of elevated serum creatine kinase (CK) levels and were found to have a dystrophinopathy. The molecular defect consisted of a deletion of variable extent within the central rod domain of the dystrophin gene, involving either exons 32-44 or 48-51 or 48-53. In each family we found the same deletion in at least one adult male relative aged from 40 to 77 years, who was either completely asymptomatic or had very mild muscle involvement (thin muscles and/or mild scoliosis), with normal or borderline CK levels. This study suggests once again that deletions of the central rod domain of dystrophin may be associated with elevation of serum CK as the only manifestation and that prediction of the clinical severity based solely on the molecular findings should be interpreted with caution.

Adult↗
PubMed1997

Molecular diagnosis and carrier screening for beta thalassemia.

Thalassemias are common autosomal recessive disorders especially in populations of Mediterranean, Middle Eastern, and Far Eastern descent. Relatively high incidence is also observed in people of Asian Indian origin but the incidence is more limited in those of African descent. Beta Thalassemias are heterogeneous at the molecular level, with more than 150 different molecular defects identified to date. Despite this heterogeneity, each at-risk population has its own spectrum of common mutations, usually from 5 to 10, a finding that simplifies mutation analysis. Homozygosity for beta thalassemias usually results in transfusion-dependent thalassemia major and, rarely, in mild non-transfusion-dependent conditions. Molecular diagnosis may be used to define genotypes associated with mild forms. Advances in carrier diagnosis using hematologic analysis followed by mutation analysis have made possible the population screening of women at childbearing age and prenatal diagnosis. This approach in combination with nondirective genetic counseling has resulted in a consistent decline of the birth of affected homozygotes in several Mediterranean at-risk populations, as well as knowledge of the risks of being a carrier. Molecular diagnosis of homozygotes and identification of carriers of beta thalassemia may lead to improved clinical management of patients with the disorder and prevention of the birth of affected homozygotes.

DNA Mutational Analysis↗
PubMed1997

Hyperbilirubinaemia in heterozygous beta-thalassaemia is related to co-inherited Gilbert's syndrome.

The reasons why heterozygotes for beta-thalassaemia have considerable variation in serum bilirubin levels are unknown. High levels of bilirubin could be related to the co-inherited Gilbert's syndrome, determined either by mutations of the coding region or by variation in the A(TA)nTAA motif of the promoter of the bilirubin UDP-glucuronosyltransferase gene (UGT-1). We sequenced the coding and the promoter region of UGT-1A or characterized the A(TA)nTAA motif of the promoter by denaturing gel electrophoresis of radioactive amplified products. The results were correlated with bilirubin levels in 49 beta-thalassaemia heterozygotes for codon 39 (CAG --> TAG) nonsense mutation. 21 normal individuals and 32 unrelated patients with Gilbert's syndrome served as controls. The coding sequence region of the UGT-1A was normal. Five beta-thalassaemia heterozygotes, who were homozygous for the extra (TA) bases in the A(TA)nTAA element of the promoter of UGT-1A, the configuration present in homozygosity in Gilbert's syndrome, had higher bilirubin levels compared to those with the (TA)6/(TA)7 or (TA)6/(TA)6 configurations. In the group of 32 patients with Gilbert's syndrome, 31 of whom had the (TA)7/(TA)7 configuration, we detected 14 heterozygotes for beta-thalassaemia, a figure much higher than predicted on the basis of the carrier rate. Homozygosity for the (TA)7 motif, the typical promoter configuration of Gilbert's syndrome, is one of the factors determining hyperbilirubinaemia in heterozygous beta-thalassaemia.

Genotype↗
PubMed1997

Hb Puttelange [beta 140(H19)Ala-->Val] in an Italian man with polycythemia.

Hb Puttelange [beta 140(H18)Ala-->Val] was found in a 51-year-old Italian man who had mild polycythemia. The variant eluted from ion exchange high performance liquid chromatography at a position between Hb A and Hb A2. It comprised approximately 34% of the total hemoglobin, was weakly unstable and exhibited an increased oxygen affinity. Amplification of the beta-globin exons and nucleotide sequencing revealed a heterozygosity for a GCC-->GTC mutation in codon 140 corresponding to an Ala-->Val replacement. This substitution accounts for the altered functional properties, probably by producing indirect perturbation of the 2 3-diphosphoglycerate pocket through the nearby lysine residue at beta 82(EF6).

Chromatography, High Pressure Liquid↗
PubMed1996

Control of beta-thalassaemia by carrier screening, genetic counselling and prenatal diagnosis: the Sardinian experience.

Homozygous beta-thalassaemia in a number of at-risk populations (Greek and Turkish Cypriots, Greeks, Continental Italians and Sardinians) has been prevented at the population level by programmes based on carrier screening, genetic counselling and prenatal diagnosis. The Sardinian experience is based on a 20-year programme. Voluntary screening has been offered to prospective parents and, primarily, to women with an ongoing pregnancy. Education of the population at large, training of health personnel, and use of posters and informative booklets have been critical elements for the success of the programme. Genetic counselling has been carried out in a non-directive manner following well-established guidelines. The use of extended family screening magnified the efficacy of the screening programme, allowing the identification of the large majority of parents at risk by screening only 13% of the population at child-bearing age. Following counselling, the large majority of parents accepted prenatal diagnosis. Definition of the parents' mutation and prenatal diagnosis were carried out by a number of PCR-based procedures. The programme was effective, as indicated by the reduction of the birth rate of thalassaemia major from 1:250 live births to 1:4000.

Genetic Counseling↗
PubMed1996

Simultaneous automated determination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in whole blood.

We report a potentiometric fully automated method for determining red cell glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities and the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index using 25 microliters of whole blood. No sample pre-treatment (e.g., preparation of the haemolysate) is needed and the measurements are performed at pH 8.0 and 37 degrees C under the conditions recommended by the ICSH committee. The reproducibility was constantly good, with within-run CV of 1.0% (glucose 6-phosphate dehydrogenase) and 5.9% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for activities in glucose 6-phosphate dehydrogenase non-deficient adults, and of 2.3% (glucose 6-phosphate dehydrogenase, G6PD) and 2.5% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for G6PDMediterranean heterozygotes. Linearity was observed up to an activity of 2800 U/l of glucose 6-phosphate dehydrogenase. Results of glucose 6-phosphate dehydrogenase activity (U/l) in whole blood (y) correlated well with those obtained with the previously described monostarter assay, performed at pH 9.2 (y = 0.60x + 37; n = 80; r = 0.991). Results of 6-phosphogluconate dehydrogenase (U/l) in whole blood (y) correlated well with those obtained by the ICSH recommended method (x) (y = 0.779x - 44; n = 23; r = 0.991). Reference intervals are reported for glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index relatively to normal, beta- and alpha-thalassaemic glucose 6-phosphate dehydrogenase non-deficient adults, to glucose 6-phosphate dehydrogenase deficient adult males and to G6PDMediterranean non-thalassaemic heterozygotes. We demonstrate that the diagnostic sensitivity of the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index in detecting the G6PDMediterranean heterozygotes is superior to that of the glucose 6-phosphate dehydrogenase activity alone.

Adolescent↗
PubMed1995

Variability of the immunoglobulin heavy chain constant region locus: a population study.

The Immunoglobulin Heavy chain Constant region (IGHC) locus is a multigene family composed of highly homologous segments often involved in unequal crossings over that lead to deleted and duplicated haplotypes. The frequencies of these haplotypes in 558 individuals from Lombardy, Veneto, Puglia and Sardinia were determined by Pulsed Field Gel Electrophoresis (PFGE), followed by Southern blotting with four IGHC probes, and compared with those observed in 110 subjects from Piedmont. Twenty deletions and 60 duplications were characterized, all in heterozygous individuals except for 2 homozygous deletions. The differences in frequency between the five populations were not significant. The deletions/duplications involved one or more genes: GP-A2, A1-E and G4 duplications, and A1-E and GP-A2 deletions were the most common. Four new duplications are described: three, involving the genes from GP to A2, from G2 to G4, and G4, are counterparts of known deletions. The fourth duplication spans from GP to G2. A G1 deleted heterozygous individual never previously described in Italy is reported. All the rearranged haplotypes seem to be the result of unequal crossing over. The difference between the number of duplications and deletions was significant in Sardinia, Lombardy, Puglia and in the total of 668 subjects (P < 0.001). This may be due to selection or genetic drift.

Blotting, Southern↗
PubMed1995

A promoter mutation, C-->T at position -92, leading to silent beta-thalassaemia.

This study describes the clinical phenotype of the C-->T mutation at position -92 of the beta-globin gene. Excluding two cases with HbA2 levels within the range of the beta-thalassemia carrier state, heterozygotes for this mutation showed normal or borderline red blood cells count, Hb levels, MCV, MCH and HbA2 values, and unbalanced globin chain synthesis. Compound heterozygotes for the -92 C-->T mutation and a beta zero-thalassaemia mutation (beta zero 39) (two cases) or severe beta+-thalassaemia (beta+ IVSII nt 745) (two cases) developed thalassaemia intermedia. According to these characteristics, the -92 promoter mutation should be added to the list of silent beta-thalassaemias.

Base Sequence↗