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We describe a new hemoglobin (Hb) variant, found in a 6-year-old Italian male living in Pistoia, Italy. An abnormal pattern compatible with a Hb A2 variant was observed on capillary electrophoresis...

Negli ultimi anni il ruolo del Laboratorio (di 1° livello o di livello intermedio), dedicato alla diagnostica dei difetti dell’emoglobina, sta diventando sempre piu centrale e impegnativo a causa dell’aumentato numero di difetti che possono essere osservati in una popolazione sempre piu eterogenea. Gli esami per le emoglobinopatie a volte richiesti con modalita non appropriate rispetto alle raccomandazioni specifiche delle Societa scientifiche necessitano di un percorso diagnostico appropriato, che comporta l’utilizzo di piu strumenti, con principi di funzionamento diversi, tali da fornire informazioni utili e suggerire percorsi di diagnosi molecolare piu mirati. Ne e un esempio il caso di una rara variante emoglobinica, identificata come Hb Hekinan.

The increase in haemoglobin (Hb)A(2) level is the most significant parameter in the identification of beta thalassaemia carriers. However, in some cases the level of HbA(2) is not typically elevated and some difficulties may arise in making the diagnosis. For these reasons the quantification of HbA(2) has to be performed with great accuracy and the results must be interpreted together with other haematological and biochemical evidence. The present document includes comments on the need for accuracy and standardisation, and on the interpretation of the HbA(2) value, reviewing the most crucial aspects related to this test. A practical flow-chart is presented to summarise the significance of HbA(2) estimation in different thalassaemia syndromes and related haemoglobinopathies.

In situ high temperature single crystal X-ray diffraction (XRD) experiments have been performed on a chemically quasi-ideal omphacite sample up to 1000°C. The lattice parameters were studied as a function of temperature, and their thermal expansion coefficients determined. The b and c cell edges show discontinuities as a function of temperature which are interpreted here in terms of intracrystalline cation diffusion processes. Structure refinements have been carried out using data collected at room temperature, at 800°C and at ambient conditions after cooling. The structural behaviour as a function of temperature of chemically quasi-ideal omphacites is compared with those of jadeite and diopside.

Aims To suggest the possible unreliability of HbA1c determination in presence of haemoglobin variants during routine metabolic evaluation. Methods We present a case of Camperdown haemoglobin, accidentally detected in a middle-aged Italian man during routine metabolic evaluation for newly diagnosed diabetes. The haemoglobin variant has been identified by exchange high performance liquid chromatography (CE-HPLC) (VARIANT™ HbA1c Program, Bio-Rad Laboratories, Hercules, CA, USA), and characterized at molecular level by direct sequencing. Results A 56-year-old male of Northern Italian origin, presented to our centre for a Type 2 diabetes mellitus of recent diagnosis. HbA1c determination was routinely determinated. The patient's chromatogram showed an inappropriate peak of 38.5% in the HbA1c position suggestive for the presence of abnormal haemoglobin. Further evaluation identified an abnormal haemoglobin peak even higher (49.5%) eluting at 1.34 minutes in P2-window. Molecular characterization of the mutation showed a nucleotide replacement, AGG AGC at codon 104, causing the amino acid replacement Arg Ser at position 104 (G6) that give rise to Hb Camperdown. Conclusions Haemoglobinopathies can lead to inaccurate glycated haemoglobin level determination. In patients carrying haemoglobin variants, the different methods for determinations of glycated haemoglobin could result in different errors, showing either higher or lower values than expected.

Hemoglobin Dallas, an α-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity ( p 1 2 = 1 mmHg at pH 7.3 and 20°C), diminished cooperativity (0n, the Hill coefficient = 1.7 ) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20°C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be atttributed to a more ‘relaxed’ T-state.

Hb Abruzzo was discovered in 1972 by Tentori et al. (1,2) in two brothers from a village in Abruzzo. Both had the compound heterozygosity of this variant and an apparent β o -thalassemia, and suffered from a mild hemolytic anemia, splenomegaly, and erythrocytosis. We recently observed the same variant in a second Italian family, also in association with a β o -thal. The family comes from the village of Troia (Foggia) which is located in the region known as Puglia, and was first studied at the Transfusional Center of Monza Hospital (Milan)

To suggest the possible unreliability of HbA(1c) determination in presence of haemoglobin variants during routine metabolic evaluation.We present a case of Camperdown haemoglobin, accidentally detected in a middle-aged Italian man during routine metabolic evaluation for newly diagnosed diabetes. The haemoglobin variant has been identified by exchange high performance liquid chromatography (CE-HPLC) (VARIANT trade mark HbA(1c) Program, Bio-Rad Laboratories, Hercules, CA, USA), and characterized at molecular level by direct sequencing.A 56-year-old male of Northern Italian origin, presented to our centre for a Type 2 diabetes mellitus of recent diagnosis. HbA(1c) determination was routinely determinated. The patient's chromatogram showed an inappropriate peak of 38.5% in the HbA(1c) position suggestive for the presence of abnormal haemoglobin. Further evaluation identified an abnormal haemoglobin peak even higher (49.5%) eluting at 1.34 minutes in P2-window. Molecular characterization of the mutation showed a nucleotide replacement, AGG --AGC at codon 104, causing the amino acid replacement Arg --Ser at position 104 (G6) that give rise to Hb Camperdown.Haemoglobinopathies can lead to inaccurate glycated haemoglobin level determination. In patients carrying haemoglobin variants, the different methods for determinations of glycated haemoglobin could result in different errors, showing either higher or lower values than expected.

A young woman aged 21 was found to be a new carrier of Hb-Belfast: beta 15 (A 12) Trp--Arg, and the characteristics of her hemoglobinopathy were not different from those of the four cases so far described: mild hemolysis with molecular instability of the abnormal Hb, red cells inclusion bodies, and slight alterations of some functional parameters of whole blood. On this occasion, direct DNA analysis indicated the genomic nucleotide replacement of the disease: TGG-AGG. This was inherited by the mother, originating from Bari (Apulia).

Hb Camperdown was discovered in 1975 in a Maltese family living in Australia (1). This slightly unstable variant, which can be separated from Hb A by various electrophoretic and chromatographic techniques, constituted nearly 50% of the hemoglobin (Hb) in the heterozygote. Here we describe the characterization of the same variant found in two unrelated males living in Northern Italy

An electrophoretically slow-moving hemoglobin, with abnormal beta chains, has been found in a young woman and in three members of her family. This variant amounted to 41% of the total Hb, and did not cause important clinical manifestations, although characterized by decreased oxygen affinity. Structural and aminoacid analyses revealed the mutation of Hb-Agenogi: 90 (F6) Glu--Lys, a rare variant so far detected in unrelated racial and ethnic groups. This is the first affected family of ascertained Piedmontese ancestry.

Hb Matera is a newly discovered, slow-moving, beta chain variant observed in four members of an Italian family. The variant had a decreased stability in heat and isopropanol tests and numerous red cells contained inclusion bodies. Structural analysis revealed that methionine at position beta 55 was replaced by lysine. Hb Matera's instability is due to an interference in the alpha 1-beta 1 contact of the hemoglobin molecule.

An uncommon abnormal hemoglobin, Hb Belfast (R 15 Trp - Arg) was discovered in a patient of Italian origin. The patient was a 42-year-old man who developed two episodes of jaundice after a prolonged administration of an antituberculous drugs. Family studies revealed that several members were asymptomatic carriers of Hb Belfast.

In the series of cubic phases, described by the general formula Ln303+Ln(An)64+Li24O69, recently reported, the member belonging to the La-Th-Li-O system was submitted to X-ray single crystal analysis. The structure was solved and refined on the basis of 960 unique diffractometer reflections at room temperature (Mo Ka radiation) in space group Pm3n with a 0 = 12.137(1) A ; Z = 36; μ = 290 cm−1; Rw=0.020. A detailed description of the structure is given; some relationships with other cubic structures in the La-Li-Me-O systems (Me = Fe,Pt,Ti,Cr,Ga) are also indicated.

A survey of hemoglobinopathies in northern Sardinia revealed a high frequency (0.3%) of carriers of a hematologic condition characterized by increased expression of fetal hemoglobin during adult life (hereditary persistence of fetal hemoglobin or HPFH). In spite of a normal hematologic phenotype, the heterozygous carriers for this condition display about 12% HbF, almost exclusively of the Aγ type; compound heterozygotes with β-thalassemia have 20%–26% HbF and run a very mild clinical course. The sequence analysis of the cloned Aγ gene linked to the HPFH determinant revealed the presence of a G→A substitution at position-117 of the Aγ- gene promoter; the same mutation occurs also in Greek HPFH, although associated with different restriction polymorphisms. Another hereditary condition characterized by increased HbF (α2 Aγ2) level and a mild thalassemic phenotype in Sardinia is associated with the-196 C→T substitution in the Aγ-globin gene promoter (Sardinian δβ-thalassemia). Population studies using oligonucleotides complementary both to the-117 G→A and-196 C→T mutations and the corresponding normal sequences confirm the presence of these mutations only in HPFH and δβ-thalassemia chromosomes and exclude these changes being common DNA polymorphisms.

The new triclinic phase Ca5(HAsO4)2(AsO4)2 · 5 H2O, polymorph of monoclinic vladimirite, was obtained by dehydration of Ca5(HAsO4)2(AsO4)2 · 9H2O (ferrarisite) at 60°C. Unit-cell data are: a = 8.286(4), b = 6.673(3), c = 9.743(4) Å, α = 86.58(3), β = 111.10(3), γ = 99.74(3)°; Z = 1, space group P[unk]. The structure was solved by direct methods and least-squares refined to R = 0.088, using 1200 observed counter reflections (MoKα radiation). One Ca atom and three water molecules are disordered over centrosymmetrically-related positions. Isostructural (001) layers of Ca and As coordination polyhedra are observed in the structures of both nona- and penta-hydrated phases; however, they differ by a relative shift and sandwich ordered and disordered Ca octahedra, respectively, in the two cases. Dehydration of ferrarisite involves the loss of inter-layer water molecules alternatively from either set of centrosymmetrically-related positions, while layers slide and approach one another. The sandwiched Ca atoms are left attached alternatively to either side of facing layers, and replace their broken coordination bonds by new ones on the side of the shifted layer. This random character of the dehydration reaction is the cause of disorder in the penta-hydrated phase. Crystal-chemical properties of the M5(HAsO4)2(AsO4)2 · n H2O phases are analyzed.

Mg3(P04h' 22 H20-II crystallizes in space group P f, with a 6.937(3),b = 6.932(3), c = 16.132(5)A, a = 82.15(3), f3 = 89.72(3), y 119.49(3) , Z = 1. The structure was solved (direct methods) and refined anisotropically (least squares) to R = 0.042, using 1826 observed reflections measured on a single-crystal diffractometer (MoKa radiation). All hydrogen atoms were located from difference maps and included in the refinement. (001)layers formed by Mg(H20)6 and P04 polyhedra and by lattice water moleculesare linked by hydrogen bonds and stacked with the. . . A BB' A. . . scheme.A structure previously studied by other authors has been recognized as a polytype, Mg3(P04h . 22 H20-I, where the pair of layers AB is nearly identical as in II, but the B' layer is oriented differently with respect to B. The two orientations are related by a mirror pseudo-symmetry of the B layer. Differencesof the hydrogen bonding scheme in the two cases are pointed out.

Hb Contaldo with a HIS→ARG substitution at position 103(G10) of the α chain is a newly discovered unstable Hb variant observed in an Italian child. Its instability is probably due to the disruption of the hydrogen bond between α103(G10)His and β108(G10)Asn. The structural variation in the core segment was determined through analysis of tryptic peptides from digests of the αx and oxidized αx (with performic acid) chains, which were separated by HPLC. Similar analyses were made for the αX chain of the rare Hb Manitoba in which α102(G9) Ser is replaced by Arg. This variant was observed for the first time in an Italian patient, and was also studied in a member of a previously described Canadian family.

Hb Contaldo with a His----Arg substitution at position 103(G10) of the alpha chain is a newly discovered unstable Hb variant observed in an Italian child. Its instability is probably due to the disruption of the hydrogen bond between alpha 103(G10)His and beta 108(G10)Asn. The structural variation in the core segment was determined through analysis of tryptic peptides from digests of the alpha X and oxidized alpha X (with performic acid) chains, which were separated by HPLC. Similar analyses were made for the alpha X chain of the rare Hb Manitoba in which alpha 102(G9) Ser is replaced by Arg. This variant was observed for the first time in an Italian patient, and was also studied in a member of a previously described Canadian family.

An uncommon abnormal hemoglobin, Hb Belfast (beta 15 Trp replaced by Arg) was discovered in a patient of Italian origin. The patient was a 42-year-old man who developed two episodes of jaundice after a prolonged administration of an antituberculous drugs. Family studies revealed that several members were asymptomatic carriers of Hb Belfast.

In our researches on the molecular meccanisms of cellular adhesion and density dependence inhibition we observed the SGS-3A neoplastic cells show a cell-cell adhesion higher than the homogenetic fibroblasts. This adhesion increases in single cells prepared with EDTA rather than Trypsin. Specific and non specific intercellular adhesion assays demonstrate that ligand molecules of coat of neoplastic cells are strongly trypsin-sensitive; on the contrary ligand molecules of coat of fibroblasts are more trypsin-resistent.

The agar suspension culture method was applied to enrich for neoplastic cells in cultures of a cellular strain (SGS-3) derived from Sarcoma Galliera. This method suppresses the growth of normal cells without impairing colony formation by neoplastic cells. The analysis by electron microscopy of colonies of SGS-3A cells cultured in agar medium shows that cytoplasmatic bridges are present in the regions of cell-cell contact.