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Studies investigating the potential role of circulating bile acids (BAs) as diagnostic biomarkers for cholangiocarcinoma (CCA) are sparse and existing data do not adjust for confounding variables. Furthermore, the mechanism by which BAs affect the expression of the oncogenic mucin 5AC (MUC5AC) has never been investigated. We performed a case-control study to characterise the profile of circulating BAs in patients with CCA ( n = 68) and benign biliary disease (BBD, n = 48) with a validated liquid chromatography-tandem mass spectrometry technique. Odd ratios (OR) for CCA associations were calculated with multivariable logistic regression models based on a directed acyclic graph structure learning algorithm. The most promising BAs were then tested in an in vitro study to investigate their interplay in modulating MUC5AC expression. The total concentration of BAs was markedly higher in patients with CCA compared with BBD controls and accompanied by a shift in BAs profile toward a higher proportion of primary conjugated BAs (OR = 1.50, CI: 1.14 to 1.96, p = 0.003), especially taurochenodeoxycholic acid (TCDCA, OR = 42.29, CI: 3.54 to 504.63, p = 0.003) after multiple adjustments. Western blot analysis of secreted MUC5AC in human primary cholangiocytes treated with primary conjugated BAs or with TCDCA alone allowed us to identify a novel 230 kDa isoform, possibly representing a post-translationally modified MUC5AC specie.

Chromium(II)-based extended metal atom chains have been the focus of considerable discussion regarding their symmetric versus unsymmetric structure and magnetism. We have now investigated four complexes of this class, namely, [Cr 3 (dpa) 4 X 2 ] and [Cr 5 (tpda) 4 X 2 ] with X = Cl - and SCN - [Hdpa = dipyridin-2-yl-amine; H 2 tpda = N 2 , N 6 -di(pyridin-2-yl)pyridine-2,6-diamine]. By dc/ac magnetic techniques and EPR spectroscopy, we found that all these complexes have easy-axis anisotropies of comparable magnitude in their S = 2 ground state (| D | = 1.5-1.8 cm -1 ) and behave as single-molecule magnets at low T . Ligand-field and DFT/CASSCF calculations were used to explain the similar magnetic properties of tri- versus pentachromium(II) strings, in spite of their different geometrical preferences and electronic structure. For both X ligands, the ground structure is unsymmetric in the pentachromium(II) species (i.e., with an alternation of long and short Cr-Cr distances) but is symmetric in their shorter congeners. Analysis of the electronic structure using quasi-restricted molecular orbitals (QROs) showed that the four unpaired electrons in Cr 5 species are largely localized in four 3d-like QROs centered on the terminal, "isolated" Cr 2+ ion. In Cr 3 complexes, they occupy four nonbonding combinations of 3d-like orbitals centered only on the two terminal metals. In both cases, then, QRO eigenvalues closely mirror the 3d-level pattern of the terminal ions, whose coordination environment remains quite similar irrespective of chain length. We conclude that the extent of unpaired-electron delocalization has little impact on the magnetic anisotropy of these wire-like molecular species.

The stringlike complex [Fe 4 (tpda) 3 Cl 2 ] (2; H 2 tpda = N 2 , N 6 -bis(pyridin-2-yl)pyridine-2,6-diamine) was obtained as the first homometallic extended metal atom chain based on iron(II) and oligo-α-pyridylamido ligands. The synthesis was performed under strictly anaerobic and anhydrous conditions using dimesityliron, [Fe 2 (Mes) 4 ] (1; HMes = mesitylene), as both an iron source and a deprotonating agent for H 2 tpda. The four lined-up iron(II) ions in the structure of 2 (Fe···Fe = 2.94-2.99 Å, Fe···Fe···Fe = 171.7-168.8°) are wrapped by three doubly deprotonated twisted ligands, and the chain is capped at its termini by two chloride ions. The spectroscopic and electronic properties of 2 were investigated in dichloromethane by UV-vis-NIR absorption spectroscopy, 1 H NMR spectroscopy, and cyclic voltammetry. The electrochemical measurements showed four fully resolved, quasi-reversible one-electron-redox processes, implying that 2 can adopt five oxidation states in a potential window of only 0.8 V. Direct current (dc) magnetic measurements indicate dominant ferromagnetic coupling at room temperature, although the ground state is only weakly magnetic. On the basis of density functional theory and angular overlap model calculations, this magnetic behavior was explained as being due to two pairs of ferromagnetically coupled iron(II) ions ( J = -21 cm -1 using JŜ i ·Ŝ j convention) weakly antiferromagnetically coupled with each other. Alternating-current susceptibility data in the presence of a 2 kOe dc field and at frequencies up to 1.5 kHz revealed the onset of slow magnetic relaxation below 2.8 K, with the estimated energy barrier U eff / k B = 10.1(1.3) K.

The structure of pentachromium(ii) extended metal atom chain [Cr 5 (tpda) 4 Cl 2 ] (2), which behaves as a single molecule magnet at low temperature, was investigated by Density Functional Theory (DFT) calculations and spectroscopic studies without the constraints of a crystal lattice (H 2 tpda = N 2 ,N 6 -bis(pyridin-2-yl)pyridine-2,6-diamine). DFT studies both in the gas phase and including CH 2 Cl 2 solvent effects indicate that an unsymmetric structure (C 4 point group), with pairs of formally quadruply-bonded metal ions and one terminal metal center, is slightly more stable (2.9 and 3.9 kcal mol -1 ) than a symmetric structure (D 4 point group). Isotopically-labelled samples (2-d 8 and 2-d 16 ) have then been prepared to aid in molecular symmetry determination by combined 1 H and 2 H NMR studies in dichloromethane solution. The spectra are strongly suggestive of a symmetric (D 4 ) framework, indicating fast shuttling between the two unsymmetric forms over the timescale of NMR experiments. Procedures for a high-yield Pd-free synthesis of H 2 tpda and for site-selective post-synthetic H/D exchange of aromatic H 2 tpda hydrogens are also reported.

A twin pair affected by juvenile myelomonocytic leukemia (JMML) with the same somatic PTPN11 mutation and abnormal chromosome 7 in bone marrow samples but distinct prognostic gene expression signatures, received a matched-unrelated donor and matched-unrelated cord blood transplant, respectively. Both twins fully engrafted, but after 6 months, the twin with an acute-myeloid-like (AML-like) signature at diagnosis rejected the graft and had an autologous reconstitution. A second transplant with an unrelated 5/6-HLA-matched-loci cord blood performed after 4 months from rejection was unsuccessful. After 25 months from diagnosis, the twin with the AML-like gene expression signature died of liver failure while on progression of his JMML. The other twin, who had a non-acute-myeloid-like (non-AML-like) gene expression signature at diagnosis is in complete hematological remission with full donor chimera. This observation suggests a biological diversity of JMML also in patients with a common genetic background.

P-selectin is an adhesion molecule involved in the pathogenesis of inflammation, thrombosis, and oncogenesis. In this study of 51 polymorphisms in candidate genes for cardiovascular disease in 1561 individuals, we identified a new allelic variant of the SELP gene, g.18196_20704del, that determined the lack of genotyping for one polymorphism in one individual. It is a deletion of 2509 nucleotides which starts in intron 6 and ends in intron 8. Re-genotyping of 1023 apparent homozygotes indicated an overall allele frequency of 0.27%. The inclusion of this allelic variant in genetic association studies will avoid genotyping errors and marginally improve the sensitivity.

Polymorphisms of the human Delta-5 (FADS1) and Delta-6 (FADS2) desaturase genes have been recently described to be associated with the level of several long-chain n-3 and n-6 polyunsaturated fatty acids (PUFAs) in serum phospholipids. We have genotyped 13 single nucleotide polymorphisms (SNPs) located on the FADS1-FADS2-FADS3 gene cluster (chromosome 11q12-13.1) in 658 Italian adults (78% males; mean age 59.7 +/- 11.1 years) participating in the Verona Heart Project. Polymorphisms and statistically inferred haplotypes showed a strong association with arachidonic acid (C20:4n-6) levels in serum phospholipids and in erythrocyte cell membranes (rs174545 adjusted P value for multiple tests, P < 0.0001 and P < 0.0001, respectively). Other significant associations were observed for linoleic (C18:2n-6), alpha-linolenic (C18:3n-3) and eicosadienoic (C20:2n-6) acids. Minor allele homozygotes and heterozygotes were associated to higher levels of linoleic, alpha-linolenic, eicosadienoic and lower levels of arachidonic acid. No significant association was observed for stearidonic (C18:4n-3), eicosapentaenoic (C20:5n-3) and docosahexaenoic (C22:6n-3) acids levels. The observed strong association of FADS gene polymorphisms with the levels of arachidonic acid, which is a precursor of molecules involved in inflammation and immunity processes, suggests that SNPs of the FADS1 and FADS2 gene region are worth studying in diseases related to inflammatory conditions or alterations in the concentration of PUFAs.

Background: Several genome scans have reported linkage of markers on chromosome 7p with asthma and related phenotypes in different populations. A fine mapping in Finnish and French-Canadian populations has associated the GPR154 gene (also known as G-protein-coupled receptor for asthma susceptibility, GPRA) with elevated IgE or asthma., Objective: To confirm chromosome 7p linkage and candidate gene association in Italian families with atopic asthma., Methods: In a two-phase approach, we first performed a linkage analysis of chromosome 7, and then a family-based association study on the GPR154 gene for allergic asthma phenotypes in the Italian population., Results: The screening of 117 families with 19 microsatellite markers showed potential linkage for elevated IgE (P<0.002 at 22 cM from p-ter), asthma (P<0.005 at 44 cM), or atopy (P<0.005 at 54 cM). In the second phase of the present study, candidate gene GPR154, which is located in the phase one-linked region, was investigated in 211 families with seven single nucleotide polymorphisms (SNPs) that tag most haplotype variability, by the pedigree disequilibrium test. Elevated IgE levels were associated with two GPR154 gene SNPs (SNP 546333, P=0.0046; rs740 347, P=0.006), and with haplotypes in the global test (P=0.013). Haplotype analysis performed in nuclear families having at least 1 asthmatic parent showed a significant association with asthma (P=0.0173), atopy (P=0.0058), SPT (P=0.0025), and bronchial hyper reactivity (P=0.0163)., Conclusion: These results support a susceptibility locus for asthma and related phenotypes on chromosome 7, and are in agreement with recent reports suggesting that a common susceptibility factor for atopic manifestations in asthma is likely conferred by the locus containing the GPR154 gene.

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

The literature contains conflicting reports on the association of common variants of the interleukin (IL)-4 receptor alpha (IL4RA) gene with atopic asthma. The purpose of the present study was to investigate the linkage and association of several gene polymorphisms with atopic asthma in a large series of well-characterized individuals. Analysis of five polymorphisms (I50V, E375A, C406R, S478P and Q551R) of the IL4RA gene was performed in 823 individuals from 182 families with atopic asthmatic children from north-east Italy. The subjects were tested for clinical asthma, total serum IgE level, skin prick test positivity to common aeroallergens, and bronchial hyperresponsiveness to methacholine. The frequency of the polymorphisms was similar to that reported for other populations. The 375, 406, 478 and 551 polymorphisms were in linkage disequilibrium, as previously reported. No linkage or transmission disequilibrium was observed in the families between any mutation and any of the phenotypes investigated. No multipoint haplotype was associated with any phenotype. In conclusion, the IL4RA gene does not seem to play an important role in genetic predisposition to atopic asthma in the population tested.

Background: Allergic asthma is a multifactorial disease for which there is a widely assessed, although poorly understood, genetic involvement. Genome-wide screens reported evidence for linkage of allergic asthma-related phenotypes to several chromosomal locations. Markers on chromosome 19 have been linked to allergic asthma phenotypes in different populations in independent studies., Objective: The aim of this study was to perform a genetic linkage analysis on chromosome 19 to search for DNA markers linked to phenotypes related to allergic asthma., Methods: Using non-parametric multipoint linkage analysis on a total of 22 random DNA markers in 2 stages, a sample of 111 families (542 subjects) from north-eastern Italy, recruited through an asthmatic allergic proband, was investigated. Phenotypes examined were: clinical asthma, total serum elevated IgE, skin prick test positivity, bronchial hyper-responsiveness, and atopy defined as skin prick test positivity and/or elevated IgE. Simulation studies were performed to confirm the significance of the results., Results: A novel linkage of atopy and skin prick test positivity to marker D19S601 (19q13.3) was found. Modest evidence for linkage of atopy, skin prick test positivity, and IgE was also found to marker D19S591 (19p13.3). Simulation analysis for atopy gave an NPL-Z > 3.326 in 2 replicates out of 1000 (P = 0.002) for D19S601, and an NPL-Z > 2.56 in 16 replicates out of 1000 (P = 0.016) for D19S591., Conclusions: On chromosome 19, suggestive linkage of atopy and skin prick test positivity with marker D19S601 (19q13.3) and modest evidence of linkage of marker D19S591 (19p13.3) to the atopic phenotypes investigated were found. These results suggest that these regions may contain susceptibility loci associated to atopic phenotypes.

Background: Genome and chromosome screens reported DNA markers on chromosome 14 linked to allergic asthma or intermediate phenotypes in several populations., Objective: We sought to perform a linkage study on chromosome 14 and a further association study on candidate genes mapped in the region found to be linked to allergic asthma or intermediate phenotypes., Methods: The study consisted of a sample of 189 families (847 genotyped individuals) from a restricted geographic area in northeastern Italy. The subjects were characterized for the following phenotypes: allergic asthma, total serum IgE levels, skin prick test responses, and bronchial hyperresponsiveness (BHR) to methacholine. Genotyping was done with 14 DNA markers and 4 polymorphisms in the genes encoding alpha(1)-anti-trypsin and alpha(1)-antichymotrypsin (ACT)., Results: Multipoint analysis indicated a potential linkage of BHR with marker D14S617 (nonparametric linkage z score = 2.32, P =.01). Transmission disequilibrium of Thr -15Ala in the gene encoding ACT was observed with all the phenotypes investigated: allergic asthma, BHR, total IgE levels, or skin prick test responses (P =.041,.02,.0053, or.026, respectively)., Conclusion: Chromosome 14 screening and transmission disequilibrium testing on the gene encoding ACT suggest that it or a closely located gene may be involved in susceptibility to allergic asthma in the Italian population.

We investigated 116 Italian atopic families (560 individuals) for linkage with 13 DNA markers on chromosome 12. All the subjects were phenotyped for asthma, total serum IgE, bronchial hyperresponsiveness, skin-prick positivity to common aeroallergens, and atopy. A relative location map of the markers was prepared from Centre d'Etude du Polymorphisme Humain families. Affected sib pair multipoint linkage methods were used to perform the statistical analyses. We report suggestive linkage for asthma with markers on chromosome 12. The region of interest centers around marker D12S390 (maximum logarithm of odds [mlod] = 2.81; p = 0.003). These results provide additional support that asthma susceptibility factors are located on chromosome 12q.

To identify genetic factors for susceptibility to atopy and asthma in childhood, 1,083 subjects were identified, mainly from the Veneto region and Bolzano province in North-east Italy, of whom 817 were from 172 families with at least two affected people, 189 were sporadic cases, and 77 unrelated controls. All the subjects were characterized for clinical asthma (asthma), total serum IgE (IgE), skin prick test (SPT) reactivity to common aeroallergens and bronchial hyperresponsiveness (BHR) to methacoline test. Atopy was defined as SPT positivity and/or increased IgE levels. Several candidate genes were investigated, and genome-wide linkage analysis was been initiated. The high affinity IgE receptor beta chain (FcepsilonRIbeta) locus showed significant allele sharing in affected sib-pairs for BHR and for SPT positivity. Lymphotoxin alpha (Ltalpha) gene Ncol mutation showed a suggestive linkage with atopy, and the LTalphaNcol 2/2 genotype was found to be associated with increased total IgE levels in all females. No evidence for linkage or association of any phenotype to the tumour necrosis factor alpha (TNFalpha) - 308 mutation or to the interleukin 4 receptor alpha (IL-4Ralpha) Q576R mutation was found. BHR, asthma and increased IgE were found to be linked to X and Y long arm pseudoautosomal region (PAR2) markers. Initial data were also collected from linkage analysis with chromosome 12, 14, and 19, DNA markers. Non-parametric multipoint analysis provides preliminary evidence for linkage of asthma with D12S390, of atopy with D19S601, and of BHR with D14S617. These results suggest that several genetic factors contribute to different allergic asthma phenotypes in the population investigated.

Tumour necrosis factor (TNF) is a proinflammatory cytokine that increases human airway tissue responsiveness and is considered a candidate gene for asthma. Two common polymorphisms (LTalphaNcoI and TNFalpha-308) in the TNF gene complex were studied in 600 subjects from 131 Italian families with atopic asthmatic children. Skin prick test (SPT), total IgE levels, atopy (defined as increased IgE levels or SPT positivity or both), bronchial hyperresponsiveness, and clinical asthma were investigated. The observed distribution of the identical by descent alleles at the LTalphaNcoI locus was different from expected for SPT and atopy (p=0.015). The LTalphaNcoI genotype distribution for increased IgE levels was different between males and females (p=0.0011), and an association of the 2.2 genotype with increased IgE levels was observed in females (p=0.0032). The results indicate that the LTalpha gene, or a closely linked locus, is associated with atopy, and suggest a sex difference in the effect of the gene.

The allele frequency distributions in a series of Croats were analyzed for six unlinked polymorphic DNA loci: THO1, FESFPS, VWA01, APOB, D1S80, and D17S5. The allele frequencies were determined for 100 unrelated genomic DNA samples. The observed heterozygote frequencies of the loci ranged from 0.63 to 0.76; however, the the expected heterozygosity ranged from 0.68 to 0.82, with only D17S5 having a significant excess of homozygous phenotypes (p < 0.001). The excess homozygosity seen in the D17S5 system may be due to allelic drop-out and warrants further technical analysis of that system, given the uniform lack of significant deviation in the other five systems. The forensic usefulness of these systems can be measured using two different statistics: the power of discrimination and the likelihood of a coincidental match. The power of discrimination ranged from 0.85 to 0.94 for the 6 systems with the combined likelihood of a coincidental match based on these 6 systems of 1 in 3.6 million, or slightly less than the population of Croatia. A second, more conservative estimator of the likelihood of a match is based on the most common phenotype for each system. If someone had the most common phenotype for each of the 6 systems, the chance of a coincidental match would be approximately 1 in 64,000. For paternity testing the usefulness of a system is measured by the average power of exclusion or (1-power of exclusion), the random man not excluded. The average power of exclusion, based on observed heterozygosity, ranged from 0.33 to 0.53, and the average power of exclusion based on the expected heterozygosity ranged from 0.39 to 0.64. The combined average power of exclusion was 99.2% for these 6 systems, using the expected heterozygosity. Based on the results of testing these six systems, there is no significant substructuring within the southern Croatian populations, and these systems provide a useful tool for forensic, paternity, and anthropological applications.

We report the allele and genotype frequencies in Italians of eight unlinked commonly utilized polymorphic loci: HUMTH01, HUMFES/FPS, HLA-DQA1, LDLR, GYPA, HBGG, D7S8 and GC. Genomic DNA from at least 100 individuals was amplified by PCR and typed after electrophoresis (HUMTH01, HUMFES/FPS), or reverse dot-blot (HLA-DQA1 and the other five loci). The allelic frequencies determined were compared with available population data. These results are useful for population and individual identification studies.

The androgenetic origin of hydatidiform moles, due to a monospermic or dispermic mechanism, has been reported, and a possible pathogenetic relation with blighted ova suggested. To evaluate the origin of hydatidiform moles and their genetic relationship with blighted ova we investigated a series of samples, utilizing several hypervariable DNA polymorphisms by Southern blotting or PCR. Seven complete or partial hydatidiform mole and 49 blighted ovum cases were investigated. The results confirm the androgenetic origin of complete hydatidiform moles, which were always due in our sample to a monospermic mechanism. Our data exclude a relationship between hydatidiform moles and blighted ova, as in the latter a mixed paternal and maternal DNA contribution was always shown. A high incidence of chromosomal abnormalities in blighted ova was also found.

A multilocus genotype survey of 190 to 352 chromosomes was performed in Italians. Genomic DNA of five tandem repeat loci was amplified in vitro with the polymerase chain reaction. Variable number of tandem repeat (VNTR) or short tandem repeat loci investigated were: D1S80; AP0B, located in the 3' flanking region of the apolipoprotein B gene; D17S5; F8VWF, located in intron 40 of the von Willebrand factor gene; and D6S89. The repeat motif was from 2 to 70 bp. The polymerase chain reaction product size was from 100 to 1070 bp. Relative allele frequencies exhibited bimodal or complex distributions. The number of alleles detected in the sample varied from 10 for F8VWF to 20 for D1S80. The observed heterozygote frequencies of the loci ranged from 0.75 for D17S5 and F8VWF to 0.83 for D1S80, and were in accord with expected frequencies. No mutations were detected in a total of 566 meioses for the five loci studied. The most frequent genotype for all five loci combined has a frequency of 4.1 x 10(-6). In 90 parental determination cases, D1S80, AP0B, D17S5 and F8VWF gave conclusive information in 39/45 exclusions and in 21/45 attributions.