P. C. Giordano

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases.

Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic populations.

We have tested five haemoglobin (Hb) separation apparatuses, dedicated to haemoglobinopathy diagnostics. These are the four high performance liquid chromatography devices: VARIANT II™, HA 8160, G7, Ultra2 and the Capillary Electrophoresis apparatus from Sebia. In the first place, we focussed on the capacity of all apparatuses to detect the most common structural variants relevant for public health, these being HbS, HbC, HbE, HbD‐Punjab and HbO‐Arab. We then compared how the high HbA2β‐thalassaemia carriers were identified. All apparatuses were able to identify carriers of these traits with the expected sensitivity and specificity. With the primary goal of a high degree of conformity in basic diagnostics of haemoglobinopathies, we present the interpretation and the significance of the results on all apparatuses, and we comment on the unavoidable problems and solutions.

Purification and primary structure of ceratotoxin A and B, two antibacterial peptides from the female reproductive accessory glands of the medfly Ceratitis capitata (insecta:Diptera)

In the present article we report the purification and the amino acid sequence of two antibacterial peptides present in the secretion of the female reproductive accessory glands of the dipteran insect Ceratitis capitata. Both peptides consist of 29 amino acid residues, are heat stable, strongly basic and differ from each other for the substitution of two amino acids. Their primary sequence and predicted secondary structure are related to other families of peptides known to have lytic and/or antibacterial activity. We propose the name ceratotoxins (from Ceratitis) for these antibacterial peptides.

A novel polyadenylation signal mutation in the α2‐globin gene causing α thalassaemia

Summary. In a family of Indian origin we have identified a deletion of two bases at the polyadenylation signal sequence of the α2‐globin gene (AATAAA AATA). Three individuals heterozygous for this mutation display an αo‐thalassaemialike phenotype. Single‐stranded conformation analysis and automatic sequencing showed no additional mutations in either α1‐ or α2‐globin genes. A previously described polyadenylation sequence mutation (AATAAA AATAAG), αTSaudiα, causes HbH disease in homozygotes. In this study the patients heterozygous for the AATA(‐AA) mutation show a similar phenotype observed in the αTSaudiα heterozygotes. This confirms the observation that the inefficient transcriptional termination due to mutations of the polyadenylation sequence of the α2‐gene might interfere with the α1‐gene expression.

Molecular spectrum of beta-thalassemia in the Iranian Province of Hormozgan.

Prevention of β-thalassemia implies knowledge of the molecular spectrum occurring in the population at risk. This knowledge is necessary, especially when a prevention protocol is applied to a multiethnic population. For this purpose, we have recently analyzed a large population of Iranian patients living in the Province of Hormozgan in Iran, and a small group of Iranian patients living in The Netherlands. We have found a different mutation spectrum in both populations as compared to the data obtained by other authors for the Iranian regions of Tehran, Fars, Sistan Balouchestan, Bushehr, and Khouzestan. The IVS-I-5 (G → C) is the most frequent mutant in the province of Hormozgan (69%), followed by the IVS-II-1 (G → A) (9.6%), while the IVS-I-1 (G → A) was the most frequent defect found in the Iranian population sample in The Netherlands. The IVS-II-745 (C →G) mutation in cis with the 5′UTR (untranslated region) +20 (C → T) transition was observed in two unrelated, transfusion-dependent homozygotes, living in the Hormozgan Province where, in contrast with populations living in other provinces of Iran, no IVS-I-110 (G → A) or IVS-I-1 (G → A) mutations were found. We report the molecular spectra of our population samples and compare them with the mutation spectra observed in the Iranian populations by other authors. We discuss the severe phenotype of the patients homozygous for the IVS-II-745 (C → G) mutation, linked in cis to the 5′UTR +20 (C→ T) transition. Molecular analysis using commercial kits is briefly compared with denaturing gradient gel electrophoresis, emphasizing the value of a rapid method of detection for molecular defects in areas where many mutations occur.

Prevalence and molecular heterogeneity of alfa+thalassemia in two tribal populations from Andhra Pradesh, India

SummaryWe describe here the screening of a small group of apparently healthy individuals belonging to the tribal communities of Koya Dora and Konda Reddi. A remarkably high incidence of deletion and nondeletion α+ thalassemia mutants has been found with allele frequencies and distributions characteristic to each tribe. We have confirmed the strict relationship between Hb S levels and the number of α globin genes in double heterozygotes for the S gene and α thalassemia. In this population sample we did not find either heterozygous carriers of α0 thalassemia (deletion of both alpha genes in “cis”) or individuals showing hemolytic anemia due to inactivation of three α-globin genes (Hb H disease). Selection by malaria is most probably responsible for the prevalence of the various α+ thalassemia haplotypes among the two tribal populations of Andhra Pradesh.

The female reproductive accessory glands of the medfly Ceratitis capitata : antibacterial activity of the secretion fluid

Abstract Secretion from female reproductive accessory glands of the dipteran Ceratitis capitata was found to have antibacterial properties against E. coli . At least two basic polypeptides with mol. wt 15.5 and 4.7 kDa respectively, were identified as responsible for such activity. Furthermore, the 15.5 kDa protein is active against a number of Gram-positive and -negative bacterial strains. Lysozyme activity is also present in the secretion.

α-Thalassemia in The Netherlands: a heterogeneous spectrum of both deletions and point mutations

Abstract In this article we describe the molecular characterization of 104 independent α-thalassemia patients identified by hematological analysis and family studies. During the study, another six chromosomes were identified with rearrangements of the α-cluster or point mutations in the α2-globin gene, not associated with α-thalassemia, in healthy relatives of the patients. The molecular defects were established by Southern blot analysis and, if no deletions could be identified, the α-globin genes were investigated by denaturing gradient gel electrophoresis and single strand conformation analysis for the presence of point mutations. Following this strategy, we were able to identify the molecular basis of 131 independent α-thalassemia chromosomes. In two individuals, the α-thalassemia determinant could not be demonstrated at the molecular level. We identified eight different deletion and five non-deletion α-thalassemias, three rearrangements in the α-cluster, two α-chain variants, and a silent mutation in the α2-globin gene not associated with α-thalassemia. The large heterogeneity of α-thalassemia mutations seen in the Dutch population might be typical for nothern European countries where, besides the more common mutations introduced by migration, a variety of sporadic mutations was also found in the autochthonous population. The screening strategy as described here, capable of identifying a wide spectrum of both deletions and point mutations, identified 98% of the α-thalassemia determinants present in 133 chromosomes.

Phenotype variability of the dominant β-thalassemia induced in four Dutch families by the rare cd121 (G→T) mutation

Abstract Eight patients who were carriers of β-thalassemia induced by the cd121 (G→T) mutation are described in four nonrelated Dutch families. This mutant, which is considered rare and inherited in a dominant manner, is expressed in a different way among each of the four families and even among carriers of the same family. The symptoms vary from an hemolytic anemia of intermediate gravity with hepatosplenomegaly, inclusion bodies and erythroblastosis, to a mild anemia with minor hematological abnormalities. We report the analytical procedures used for the detection of the mutant, the hematological and clinical data of the four families and discuss the variable physiopathology of this molecular defect. We also compare the variation in fetal hemoglobin expression in relation to the haplotypes of the β-gene cluster and to the different hematological conditions. The presence of this rare mutant in four nonrelated Dutch families could derive from a single mutation or from multiple events. The existence of the four mutations in three different haplotypes suggests the occurrence of at least two independent events. The presence of five abnormal hemoglobins and the β-thalassemia defect on different haplotypes at cd121 also suggests a relatively increased rate of mutations at this particular site.

The molecular spectrum of beta-thalassemia and abnormal hemoglobins in the allochthonous and autochthonous dutch population.

The prevalence at birth of hemoglobin defects in the autochthonous North-European population is low. However, the long immigration and colonial history of the Netherlands has resulted in a group of about 1–2 million ‘autochthonous‘ inhabitants, with Asian, South-European or African ancestors, in whom a moderate birth prevalence of globin gene mutations can be expected. Furthermore, at least 10% of the Dutch population consists of recent immigrants from different countries with high birth prevalence of hemoglobinopathies. Because of the endogamous partner choice, which is prevalent in this population, the risk for homozygous progeny remains elevated. At least 100,000 carriers of hemoglobinopathies of recent allochthonous origin are present in the Netherlands, and the number of homozygous children is rising. Prevention by prenatal diagnosis requires a suitable protocol and knowledge about the molecular defects present in the country. Therefore we have analyzed a large number of patients and carriers, both at the hematological and at the DNA level. Our survey revealed 47 different β-thalassemia determinants, characterized on 223 independent chromosomes from individuals of different ethnic origins. As expected, the most prevalent mutations were largely represented. The cd39 (C→T) mutation was found in 70% of the immigrants from Morocco, Sardinia and other Central-West-Mediterranean regions while the IVS-I-110 (G→A) was prevalent in the East-Mediterranean populations. The IVS-I-5 (G→C) mutation was found in 45% of the patients of Indonesian origin. We also registered 308 independent chromosomes with common structural defects (HbS, HbC, HbE, Hb Lepore, Hb Constant Spring and HbD Punjab) and 33 chromosomes with 19 different, less frequent, rare or very rare mutants. Seven structural mutants were described for the first time and published separately. Furthermore, 139 independent chromosomes with deletional and nondeletional α-thalassemia defects were characterized.