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Biomedical subjects

B Lunghi

Publications and source records attributed to B Lunghi.

At least 19 recordsLinked to original sources
PubMed2005

The factor V Glu1608Lys mutation is recurrent in familial thrombophilia.

BACKGROUND: Co-inheritance of heterozygous factor V deficiency with FV Leiden enhances the activated protein C resistance (APCR) associated with this mutation, resulting in pseudo-homozygous APCR. The role of FV deficiency in modulating thrombotic risk in this rare condition is poorly understood. METHODS AND RESULTS: We have identified in thrombophilic patients with FV deficiency a novel FV gene mutation (c. 4996G>A), predicting the Glu1608Lys substitution in the A3 domain. The heterozygous mutation was detected in three unrelated patients, two carriers of the FV Leiden mutation, and one of the FVHR2 haplotype. The Glu1608Lys change was also present in two subjects with mild FV deficiency, and absent in 200 controls. The FV1608Lys carriers showed reduced mean FV activity (42% +/- 12%) and antigen (53% +/- 18%) levels and, in Western blot analysis, reduced amounts of intact platelet FV. The restriction fragment length polymorphism (RFLP) study identified two haplotypes underlying the mutation, which suggests that it is recurrent. In heterozygous subjects the amount of FV1608Lys mRNA in white blood cells was similar to that produced by the counterpart alleles (FVWt or FVHR2). Recombinant FV1608Lys (rFV1608Lys), detected by Western blot in the conditioned medium, was indistinguishable from rFVWt and FV antigen and activity were found to be respectively 44% +/- 20% and 13% +/- 4% of rFVWt. CONCLUSIONS: Our data indicate that FVGlu1608Lys predicts a CRM (plasma)/CRMred (cell culture) FV deficiency, and may contribute to thrombophilia in carriers of FV Leiden and FVHR2 haplotype via a pseudo-homozygosity mechanism. Our findings help to define the molecular bases of FV deficiency and thrombophilia.

Case-Control Studies↗
PubMed2005

Expression of the normal factor V allele modulates the APC resistance phenotype in heterozygous carriers of the factor V Leiden mutation.

BACKGROUND: Functional defects of the protein C pathway, detectable in plasma as activated protein C (APC) resistance, are a prevalent risk factor for venous thrombosis. The factor V (FV) Leiden mutation causes APC resistance by interfering with the APC-mediated inactivation of both FVa and FVIIIa. Co-inheritance of FV Leiden and quantitative FV deficiency on different alleles, a rare condition known as pseudo-homozygous APC resistance, is associated with pronounced APC resistance and 50% reduced FV levels, because of non-expression of the non-Leiden FV allele. OBJECTIVES: The role of normal FV in modulating the APC resistance phenotype in carriers of FV Leiden was investigated in patients with pseudo-homozygous APC resistance and in model systems. PATIENTS/METHODS: Four functional plasma assays probing both components of APC resistance (susceptibility of FVa to APC and cofactor activity of FV in FVIIIa inactivation) were employed to compare seven clinically and genetically characterized FV Leiden pseudo-homozygotes to 30 relatives with different FV genotypes (including 12 FV Leiden heterozygotes and seven carriers of FV deficiency) and to 32 unrelated FV Leiden homozygotes. RESULTS AND CONCLUSIONS: All assays consistently indicated that FV Leiden pseudo-homozygotes are significantly more APC-resistant than heterozygotes and indistinguishable from homozygotes. Thrombin generation measurements in FV-deficient plasma reconstituted with purified normal FV and FV Leiden confirmed these observations and showed that the expression of the normal FV allele is an important modulator of APC resistance in FV Leiden heterozygotes. These findings provide an explanation for the higher thrombotic risk of FV Leiden pseudo-homozygotes when compared with heterozygotes.

Activated Protein C Resistance↗
PubMed2005

Different lung responses to cigarette smoke in two strains of mice sensitive to oxidants.

The development of cigarette smoke-induced pulmonary changes in C57 Bl/6J and DBA/2 mice was investigated. Both strains are sensitive to oxidants and C57Bl/6J mice are moderately deficient in serum alpha1-proteinase inhibitor. Following chronic exposure to cigarette smoke, patchy emphysema was present in mice of both strains, but developed faster in DBA/2 mice. A positive reaction for mouse neutrophil elastase was seen on the septa of both strains. Additionally, the DBA/2 mice developed a uniform parenchymal dilation that was preceded by the appearance of apoptotic cells in areas with a low signal for vascular endothelial growth factor-receptor 2. Fibrotic areas scattered throughout the parenchyma, coupled with a positive immunohistochemical reaction for transforming growth factor-beta was seen only in DBA/2 mice. Both DBA/2 and C57Bl/6J strains showed epithelial cell injury and areas of deciliation in their airways. However, the appearance of goblet cell metaplasia was common in C57Bl/6J mice but rare in DBA/2 mice. A positive immunohistochemical reaction for interleukin (IL)-4, IL-13 and MUC5AC was seen only in the airways of C57Bl/6J mice. Strain characteristics (alpha1-proteinase inhibitor levels, sensitivity to oxidants, and constitutive levels of vascular endothelial growth factor-receptor 2) and phenotypical responses (apoptosis and cytokine distribution) may condition parenchymal and airway changes to cigarette smoke.

Analysis of Variance↗
PubMed2002

Venous thromboembolism in young women; role of thrombophilic mutations and oral contraceptive use.

AIMS: The interaction between the R506Q mutation of factor V and the G20210A mutation of prothrombin with oral contraceptives on venous thromboembolism was evaluated. METHODS AND RESULTS: Three hundred and one women of reproductive age who had venous thromboembolism (140 while using oral contraceptives) and 650 healthy women (173 on oral contraceptives at presentation) were examined. Of the patients, 19.3% were carriers of R506Q (two homozygotes) and 9.6% were heterozygous carriers of G20210A; eight patients (2.7%) were heterozygous for both mutations. Among controls, 2.9% were carriers of R506Q, 3.1% of G20210A, while one case was a heterozygous carrier of both mutations. The relative risk (odds ratio) associated with carriership of R506Q or G20210A mutations was 10.3 and 4.7, respectively; it was 45.6 in carriers of both mutations. The odds ratio of using oral contraceptives in the absence of both mutations was 2.4. The odds ratios according to oral contraceptives use and the presence of R506Q or G20210A or both mutations were 41.0, 58.6 and 86.5, respectively. While the odds ratio for R506Q remains elevated (8.9) in non-oral contraceptive users, the odds ratio for G20210A was 2.0 and did not reach statistical significance. CONCLUSIONS: Our data showed a strong interaction between oral contraceptive use and the presence of either R506Q or G20210A mutations. In non-oral contraceptive users the risk of venous thromboembolism was significantly increased in carriers of R506Q but not in those with the G20210A mutation.

Adolescent↗
PubMed2001

Effects of cigarette smoke in mice with different levels of alpha(1)-proteinase inhibitor and sensitivity to oxidants.

The role of strain difference in the response to cigarette smoke was investigated in mice. Mice of the strains DBA/2 and C57BL/6J responded to acute cigarette smoke with a decrease of the antioxidant defenses of their bronchoalveolar lavage (BAL) fluids. On the other hand, under these conditions ICR mice increased their BAL antioxidant defenses. Mice of these three strains were then exposed to cigarette smoke (three cigarettes/d, 5 d/wk) for 7 mo. Lung elastin content was significantly decreased in C57BL/6J and DBA/2 but not in ICR mice. Also, emphysema, assessed morphometrically using three methods, was present in C57BL/6J and DBA/2 but not in ICR mice. In an additional study pallid mice, with a severe serum alpha(1)-proteinase inhibitor (alpha(1)-PI) deficiency and that develop spontaneous emphysema, were exposed to cigarette smoke for 4 mo. This resulted in an acceleration of the development of the spontaneous emphysema assessed with morphometrical and biochemical (lung elastin content) methods. All these results indicate that sensitivity to the effects of cigarette smoke is strain-dependent and cigarette smoke accelerates the effects of alpha(1)-PI deficiency.

Animals↗
PubMed2001

A highly polymorphic microsatellite in the factor V gene is an informative tool for the study of factor V-related disorders.

The role of factor V (FV) mutations in activated protein C (APC) resistance and FV deficiency is well established. We report on the identification of a highly polymorphic (AT)n microsatellite marker in the FV gene, which represents an informative tool for the investigation of the origin and evolution of pathologically relevant FV genetic components. A high number of different microsatellite alleles were found to be associated with FV R506Q and FV H1299R, two single-origin mutations. An example of the use of the microsatellite marker in family studies of thrombophilia and FV deficiency is also provided.

Factor V↗
PubMed2001

A missense mutation (Y1702C) in the coagulation factor V gene is a frequent cause of factor V deficiency in the Italian population.

BACKGROUND AND OBJECTIVES: Factor V (FV) deficiency is a rare bleeding disorder whose molecular bases are poorly characterized. We have recently described a FV missense mutation (Y1702C) predicting reduced FV levels in a thrombophilic patient and in a healthy individual. The aim of the present work was to assess the prevalence of the FV Y1702C mutation among subjects with FV deficiency. DESIGN AND METHODS: Carriership of the FV Y1702C mutation was tested in 8 patients with severe FV deficiency (FV:C <8%), in 16 individuals with asymptomatic partial FV deficiency (mean FV:C 38.0%, SD 11.6%) and in 9 patients with pseudo-homozygous APC-resistance (mean FV:C 46.2%, SD 3.6%). An AccI-restriction protocol was employed for rapid mutation screening. RESULTS: The FV Y1702C mutation was detected in two unrelated patients with unmeasurable FV levels (one being homozygous and the other doubly heterozygous for a still unknown mutation) and in one subject with partial FV deficiency (FV:C 30%). A striking difference in bleeding phenotype was observed between the homozygous patient and her asymptomatic brother with the same FV genotype. A multi-point FV haplotype analysis was performed in all unrelated carriers of the FV Y1702C mutation. Three haplotypes were found to underlie the mutation in different individuals, suggesting that it might have arisen independently more than once. INTERPRETATION AND CONCLUSIONS: FV Y1702C is a common cause of FV deficiency in the Italian population and might be a recurrent mutation.

Adolescent↗
PubMed2000

Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT 20210G/A) affecting the prothrombinase complex in a thrombophilic family.

The study of the molecular bases of thrombophilia in a large family with 4 symptomatic members is reported. Three thrombophilic genetic components (FV R506Q, FV H1299R, and PT 20210G/A), all affecting the activity of the prothrombinase complex, were detected alone and in combination in various family members. In addition, a newly identified missense mutation (factor V [FV] Y1702C), causing FV deficiency, was also present in the family and appeared to enhance activated protein C (APC) resistance in carriers of FV R506Q or FV H1299R by abolishing the expression of the counterpart FV allele. The relationships between complex genotypes, coagulation laboratory findings, and clinical phenotypes were analyzed in the family. All symptomatic family members were carriers of combined defects and showed APC resistance and elevated F1 + 2 values. Evidence for the causative role of the FV Y1702C mutation, which affects a residue absolutely conserved in all 3 A domains of FV, factor VIII, and ceruloplasmin, relies on (1) the absolute cosegregation between the mutation and FV deficiency, both in the family and in the general population; (2) FV antigen and immunoblot studies indicating the absence of Y1702C FV molecules in plasma of carriers of the mutation, despite normal levels of the FV Y1702C messenger RNA; and (3) molecular modeling data that support a crucial role of the mutated residue in the A domain structure. These findings help to interpret the variable penetrance of thrombosis in thrombophilic families and to define the molecular bases of FV deficiency. (Blood. 2000;96:1443-1448)

Amino Acid Sequence↗
PubMed2000

Mutations in the R2 FV gene affect the ratio between the two FV isoforms in plasma.

Molecular genetics and biochemical studies were performed in homozygotes for the R2 allele (4070G) in the factor V gene, most of them affected by coronary artery disease. Novel polymorphisms (G642T, 156Ser; T1328C, 385Met/Thr), among which a functional candidate (A6755G, 2194Asp/Gly) located in the C2 domain of FV, were identified in the R2 gene. In chromatographic studies R2 FV appeared qualitatively identical to normal FV. However, a relative increase of the more thrombogenic and more glycosylated FV isoform (FV1) was observed in plasma of 2194Gly homozygotes (mean FV1/FV2 ratio 0.71, 95% CI 0.66-0.77) as compared to R2-free controls (0.37, 95% CI 0.34-0.40). We conclude that carriership of the R2 FV gene is associated with an imbalance between the two functionally different FV isoforms, and propose that genetically determined differential glycosylation of FV could represent a novel mechanism of thrombotic disease.

Aged↗
PubMed1999

Phenotype and genotype expression in pseudohomozygous factor VLEIDEN : the need for phenotype analysis.

The presence of a DNA mutation is frequently used to define a disease or a risk state. Because DNA typing has become easy and convenient in contrast to protein characterization, it is generally assumed that a mutation if present (or not) at the DNA level will be also present (or not) in the corresponding protein. However, discrepancies between phenotype and genotype can occur. A point mutation in the coagulation factor V gene (G1691-->A, resulting in an Arg506-->Gln amino acid substitution in the factor V molecule [factor VLEIDEN], leading to activated protein C resistance) is the most common genetic risk factor for familial thrombophilia. A pseudohomozygous factor VLEIDEN phenotype would occur if a heterozygous individual for factor VLEIDEN also did not express the "normal" (non-Leiden) factor V allele. However, to date, no data have been available to confirm the presence of only the factor VLEIDEN form in the plasma of these individuals. Platelet mRNA from 2 presumed pseudohomozygous patients and their family members was isolated, the amplified partial cDNAs were sequenced or restricted, and the allelic bands were quantified. Both patients were found to be heterozygous for the G1691-->A substitution at both the DNA and mRNA levels. The presence of either the normal or mutated form of factor V in the patients' plasmas was investigated using a monoclonal antibody to factor V that recognizes an epitope located between residues 307 and 506 of the factor Va heavy chain. No normal factor V could be detected in the plasmas of the 2 propositi. The present data demonstrate absence of a correlation between genotype at position 1691 (at the DNA and mRNA levels) and the corresponding phenotype data found in the plasmas of patients with pseudohomozygous factor VLEIDEN. Overall, these data suggest the existence of heterogeneous genetic "lesions," which interfere with factor V expression, processing, secretion, and/or stability. Because the presence of the factor VLEIDEN molecule in plasma is directly related to pathology, identification and quantification of the circulating forms of factor V in plasma may be required for the diagnosis of individuals with activated protein C resistance.

Adult↗
PubMed1998

Molecular bases of pseudo-homozygous APC resistance: the compound heterozygosity for FV R506Q and a FV null mutation results in the exclusive presence of FV Leiden molecules in plasma.

Pseudo-homozygous APC resistance, the condition resulting from compound heterozygosity for FV R506Q (FV Leiden) and quantitative FV deficiency, provides a natural model to study the interaction between procoagulant and anticoagulant defects. This paper reports a complete FV characterization of a pseudo-homozygous APC resistant thrombotic patient. The expression of the patient's non-Leiden gene was found to be severely impaired both at the mRNA and protein levels. In particular, only FV Leiden molecules were detected in the patient's plasma by immunoblotting, which accounts for the observed marked APC resistance. Analysis of the FV cDNA obtained by reverse transcription of platelet RNA revealed that the mRNA of the non-Leiden gene was extremely reduced in amount. A PAC clone containing the whole FV gene was used to design primers for a complete FV exon scanning. A 2-bp insertion at nucleotide 3706 in the large exon 13 of the non-Leiden gene, predicting a frame-shift and premature termination of protein synthesis, was identified as responsible for the FV defect. Failure to find any case of pseudo-homozygous APC resistance in a large sample (6,804) of blood donors suggests that this condition is extremely rare among normal controls and that its detection is favoured by the thrombotic risk that it may confer.

Aged↗
PubMed1997

A factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype.

Factor V gene polymorphisms were investigated to detect components that may contribute to the activated protein C (APC) resistance phenotype in patients with venous thromboembolism. A specific factor V gene haplotype (HR2) was defined by six polymorphisms and its frequency was found to be similar in normal subjects coming from Italy (0.08), India (0.1), and Somalia (0.08), indicating that it was originated by ancestral mutational events. The relationship between the distribution of normalized APC ratios obtained with the functional assay and haplotype frequency was analyzed in patients heterozygous for factor V R506Q (factor V Leiden). The HR2 haplotype was significantly more frequent in patients with ratios below the 15th percentile than in those with higher ratios or in normal controls. Moreover, the study of 10 patients with APC resistance in the absence of the factor V R506Q mutation showed a 50-fold higher frequency of HR2 homozygotes. The HR2 haplotype was associated with significantly lower APC ratios both in patients with venous thromboembolism and in age- and sex-matched controls. However, the two groups showed similar HR2 haplotype frequencies. Plasma mixing experiments showed that an artificially created double heterozygote for the factor V R506Q mutation and the HR2 haplotype had an APC ratio lower than that expected for a simple R506Q heterozygote. Time-course experiments evaluating the decay of factor V in plasma showed the normal stability of the molecule encoded by the factor V gene marked by the HR2 haplotype, which ruled out the presence of a pseudo-homozygous APC resistance mechanism. Our results provide new insights into the presence of factor V genetic components other than the factor V R506Q that are able to contribute to the APC resistance phenotype in patients with venous thromboembolism.

Alleles↗
PubMed1997

Phenotypic homozygous activated protein C resistance associated with compound heterozygosity for Arg506Gln (factor V Leiden) and His1299Arg substitutions in factor V.

Two patients from two unrelated families with a history of thrombosis showed severe plasma activated protein C (APC) resistance. However, genotypic analysis demonstrated that the patients were heterozygous for factor V (FV) Leiden mutation. Coagulation studies revealed that FV clotting activity and antigen were similarly reduced at about 50% of normal in the patients. One brother of propositus A also showed the same abnormalities. Genetic analysis showed that, in addition to FV Leiden mutation in exon 10 of the FV gene (G1691A), these patients had a transition in exon 13 of the FV gene (A4070G; R2 allele) predicting His1299Arg substitution in the mature FV. Study by RT-PCR of platelet FV mRNA indicated that the mRNA produced by the FV gene, marked by the R2 allele, was reduced in amount in both pseudohomozygous patients of family A. The R2 allele has previously been demonstrated to be significantly associated with plasma FV deficiency in the Italian population. The presence of FV deficiency did not protect the propositi from thrombosis. These data confirm that genotypic analysis is mandatory in patients with phenotypic severe APC resistance before these patients are definitely classified as homozygotes for FV Leiden and that further genotypic analysis is advisable.

Adult↗
PubMed1997

Vitamin D receptor in IMR-90 human fibroblasts and antiproliferative effect of 1,25-dihydroxyvitamin D3.

This study examines the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], serum or forskolin on the proliferation of IMR-90 fetal lung fibroblasts and demonstrates, for the first time, the presence of 1,25(OH)2D3 receptor (VDR) in this cell line. In quiescent, subconfluent cultures neither the treatment with 100 nM 1,25(OH)2D3 nor that with 50 microM forskolin influenced proliferation, while a significant increase was observed after incubation of the cells with 10% fetal bovine serum. Either cell number, determined on growing IMR-90 human fibroblasts after 48 or 72 h incubation with 100 nM 1,25(OH)2D3 or [3H]thymidine incorporation (24, 48 or 72 h incubation) significantly decreased, while protein content per cell increased. Northern blot analysis revealed the expression of the VDR gene, the VDR mRNA bands being prominent in 1,25(OH)2D3, serum or forskolin treated fibroblasts. VDR mRNA levels slightly decreased, when growing fibroblasts were exposed to 100 nM 1,25(OH)2D3 for 48 or 72 h.

Animals↗
PubMed1997

New coagulation factor V gene polymorphisms define a single and infrequent haplotype underlying the factor V Leiden mutation in Mediterranean populations and Indians.

Two novel polymorphisms were identified in the factor V gene by direct sequencing of intronic areas. One of them, located in intron 9, is the marker closest to the Leiden mutation ever described, whereas the other, in intron 16, displays a rare allele invariantly associated to the mutation. Allele-specific amplification protocols were designed to perform extensive screenings for both polymorphic sites. The new markers were used in combination with six previously described polymorphisms to define specific factor V gene haplotypes. Haplotype investigations in 506Q homozygous thrombotic patients and normal controls showed the presence of a single haplotype underlying the factor V Leiden mutation in Mediterranean populations (among which Greek Cypriots, where the R506Q mutation is particularly frequent) and Indians. When traced in the absence of the Leiden mutation, the background haplotype was found to be present and roughly as frequent as the mutation itself in these populations. These findings indicate a single mutational event, that probably occurred outside Europe, as the cause of the Leiden mutation and provide a powerful tool to investigate its evolutionary history.

Cyprus↗