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L Baronciani

Publications and source records attributed to L Baronciani.

At least 19 recordsLinked to original sources
PubMed2006

Biochemical characterization of a recombinant von Willebrand factor (VWF) with combined type 2B and type 1 defects in the VWF gene in two patients with a type 2A phenotype of von Willebrand disease.

BACKGROUND: In a patient previously diagnosed with type 2A von Willebrand disease (VWD) [absence of high and intermediate molecular weight von Willebrand factor (VWF) multimers and markedly reduced ristocetin-induced platelet aggregation (RIPA)], an infusion test of desmopressin was followed by mild thrombocytopenia. This led to further laboratory investigations of his affected brother and of family members, who showed different phenotypic patterns compatible with type 1, 2A, 2B and an uncertain classification of VWD. The two brothers were compound heterozygotes (C275R/P1337L), whereas the others members of the family were heterozygous for C275R (a novel mutation in the D1 domain) or P1337L (a type 2B mutation in the A1 domain). OBJECTIVE AND METHODS: To evaluate the role of the combined effect of the two mutations in the two brothers, C275R and P1337L recombinant (r) VWFs were transiently expressed in COS-7 cells. RESULTS: Recombinant VWF levels secreted in cell media were similar for wild-type (WT), P1337L and hybrid P1337L/WT rVWFs, reduced for hybrids C275R/P1337L and C275R/WT rVWFs, and strongly reduced for C275R rVWF. All rVWFs had a full set of multimers except C275R rVWF, which had only dimers. P1337L rVWF and C275R/P1337L rVWF showed the highest degree of binding to glycoprotein (GP) Ibalpha and the lowest to collagen, followed by P1337L/WT rVWF (with an intermediate level of binding to both ligands), and by WT rVWF with the lowest level of binding to GPIbalpha and the highest to collagen. CONCLUSION: These results suggest that the two compound heterozygous patients have a circulating VWF mainly mutated in the A1 domain (P1337L). This peculiar type 2B VWF variant showed a remarkably high affinity for the GPIbalpha platelet receptor, leading to the loss of high and intermediate molecular weight multimers and hence to decreased RIPA, as in type 2A VWD.

Collagen↗
PubMed2006

An association of candidate gene haplotypes and bleeding severity in von Willebrand disease type 2A, 2B, and 2M pedigrees.

We analyzed the association of bleeding severity with candidate gene haplotypes within pedigrees of 11 index cases of von Willebrand disease (VWD) type 2 (two type 2A, three type 2B and six type 2M), using the QTL Association model (MENDEL 5.5). In addition to the 11 index cases, these pedigrees included 47 affected and 49 unaffected relatives, as defined by VWF mutations and/or phenotype. A bleeding severity score was derived from a detailed history and adjusted for age. Donors were genotyped using a primer extension method, and eight candidate genes were selected for analysis. VWF antigen (or ristocetin cofactor activity) levels had the strongest influence on bleeding severity score. After Bonferroni correction for multiple testing, only ITGA2 promoter haplotype -52T was associated with an increased bleeding severity score (P < 0.01). This association remained statistically significant when the three type 2B pedigrees were excluded (P = 0.012) or when gender-specific bleeding categories were excluded (P < 0.01). The major haplotypes of seven other candidate genes, GP1BA, ITGA2B, ITGB3, GP6, VWF, FGB, and IL6, were not associated with bleeding severity. These results establish that genetic differences in the expression of the integrin subunit alpha2 can influence the bleeding phenotype of VWD type 2 and complement our previous findings in VWD type 1. Genetically controlled attenuation of platelet collagen receptor expression can influence risk for morbidity in clinical settings where hemostasis is compromised.

Adolescent↗
PubMed2005

Expression studies on a novel type 2B variant of the von Willebrand factor gene (R1308L) characterized by defective collagen binding.

A novel mutation, R1308L (3923G > T) was present in the heterozygous state in five members of a family with type 2B von Willebrand disease (VWD) characterized by a full set of von Willebrand factor (VWF) multimers in plasma and by the absence of thrombocytopenia before and after desmopressin (DDAVP). The defect (R1308L) was located at the same amino acid position of one of the most common mutations associated with type 2B VWD (R1308C), which is characterized by the loss of high molecular weight VWF multimers (HMWM) in plasma and the occurrence of thrombocytopenia. To understand the mechanisms of this defect, the novel (R1308L) and 'common' (R1308C) mutations were expressed in COS-7 cells, either alone or, to mimic the patients' heterozygous state, together with wild-type VWF. R1308L recombinant VWF (rVWF) had a higher affinity for the platelet glycoprotein Ibalpha (GPIbalpha) receptor than wild-type rVWF, R1308C rVWF showing an even higher affinity. A novel finding was that both mutant rVWFs showed a similarly reduced binding to collagen type I and type III in comparison with wild-type rVWF. The latter finding suggests a more important role than recognized so far for the VWF A1 domain in VWF binding to collagen, which may contribute to the in vivo hemostatic defect associated with type 2B VWD.

Adult↗
PubMed2001

Molecular characterization of the PK-LR gene in sixteen pyruvate kinase-deficient patients.

We studied the PK-LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042--1044); one four nucleotide duplication (nt 1515--1518, GGTC); one splice site [IVS6(-2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042--1044, dupl 1515--1518, IVS6(-2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042-1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515-1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(-2)t leads to an alteration of the 5' and 3' splice site consensus sequence; the cDNA analysis shows a 67-bp deletion in the first part of exon 11 (del 1437--1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.

Adult↗
PubMed2000

Molecular characterization of a multiethnic group of 21 patients with type 3 von Willebrand disease.

Type 3 von Willebrand disease is a rare autosomal disorder characterized by unmeasurable levels of von Willebrand factor and severe hemorrhagic symptoms. We studied a multiethnic group of 37 patients, from Italy (n = 14), Iran (n = 10) and India(n = 13) to identify the molecular defects and to evaluate genetic heterogeneity among these populations. Twenty-one patients (6 Italians, 9 Iranians and 6 Indians) were fully characterized at the molecular level. Twenty-four different gene alterations were identified, 20 of which have not been described previously. The majority of the mutations caused null alleles, 11 being nonsense mutations (Q218*, W222*, R365*, R373*, E644*, Q706*, S1338*, Q1346*, Y1542*, R1659*, E2129*), 4 small deletions (437delG, 2680delC, 6431delT, del 8491-8499), 3 possible splice site mutations [IVS9(-1)g-->a, IVS29(+10)c-->t, IVS40(-1)g-->c], 3 candidate missense mutations (C275S, C2174G, C2804Y), 2 small insertions (7375insC, 7921insC) and 1 large gene deletion. The latter mutation was associated with the development of alloantibodies to VWF, but this complication was also found in a patient homozygous for a nonsense mutation (Q1346*). Due to the ethnic origin of the patients most of them were the offspring of consanguineous marriages and so were homozygous for the mutations found (18/21). Our results indicate that molecular defects responsible for type 3 VWD are scattered throughout the entire VWF gene (from exon 3 to 52), and that there is no prevalent and common gene defect in the three populations studied by us.

Adolescent↗
PubMed1997

Molecular characterization of PK-LR gene in pyruvate kinase-deficient Italian patients.

We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(-2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(-2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3' untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(-2)c lead to an alteration of the 5' and 3' splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

Adolescent↗
PubMed1997

A variant of the EPB3 gene of the anti-Lepore type in hereditary spherocytosis.

The EPB3 gene encodes band 3 (anion exchanger 1) of the red cell membrane. A subset of hereditary spherocytosis (HS) is associated with EPB3 gene mutations and band 3 deficiency. We report a large Italian family in which 10 of the 27 members investigated displayed an autosomal dominant HS. SDS-PAGE revealed a reduction in band 3 in the patients. Screening of the Pst I polymorphic site confirmed the linkage of HS with the EPB3 gene. Analysis of complementary and genomic DNA showed a large additional segment. Nucleotide sequencing disclosed an in-frame duplication of 69 nucleotides (nt) including a triplet of intronic origin and a genuine exonic duplication of 66 nt. Two CCTGC sequences occurred close to one another, one near the intron 12 acceptor splice site (nt -7 to -3), and the other within exon 13 (nt 1494-1498). We assumed that the abnormal allele arose from an unequal recombination event of the anti-Lepore type between the two CCTGC sequences. At the level of the mutated protein, termed band 3 Milano, the additional segment (Gln plus duplication of residues 478-499) corresponded to the last part of the third transmembrane domain (TM3), the entire second outer loop and part of TM4 as it is currently defined in hydropathy analysis. After deglycosylation of band 3, only the normal band was detected, supporting the view that band 3 Milano is probably not incorporated into the membrane.

Adolescent↗
PubMed1996

Study of the molecular defects in glucose phosphate isomerase-deficient patients affected by chronic hemolytic anemia.

We have studied four unrelated Italian patients with chronic hemolytic anemia associated with glucose phosphate isomerase (GPI) deficiency. Using intronic primers, we were able to detect the gene alterations on the genomic DNA of the patients. Five different mutations were identified among the eight mutated alleles found: three missense mutations (301A,584T,1028G), one nonsense mutation (286T), and a four nucleotides deletion [Del 1473-IVS16(+2)]. All of these were new except for mutation 1028G, which was previously identified in a Japanese variant (GPI Narita). Two patients were homozygotes (301A/301A and 1028G/1028G), whereas the other two were compound heterozygotes sharing a common mutation [286T/584T and Del 1473-IVS16(+2)/584T]. The missense mutations were found to involve highly conserved amino acids, suggesting that these residues are crucial for the maintenance of the enzyme function. The mutation 286T results in a truncated protein of 95 amino acids in comparison with the 558 of the normal one. The four nucleotides deletion located at the junction of exon/intron 16(5'-TTGGTCGgtgagt-3') is the first GPI mutation affecting a splice site. Moreover one difference from the published sequence (473T-->G) was found in exon five in all of the eight alleles studied and in 30 normal subjects. Correlation was made between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

Adult↗
PubMed1996

Mutations in pyruvate kinase.

Pyruvate kinase (PK) deficiency due to mutations of the PKLR gene is a common cause of hereditary nonspherocytic hemolytic anemia. Thus far, 55 different mutations have been described in patients with PK-deficient hemolytic anemia. Polymorphisms within the PKLR gene and in the tightly linked glucocerebrosidase (GBA) gene suggest that PK deficiency may represent a balanced polymorphism.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1996

Interaction of ATP with erythroblast glucose-6-phosphate dehydrogenase.

ATP, complexed with magnesium and as free anion, binds to rabbit erythroblasts glucose-6-phosphate dehydrogenase (G6PD) inducing significant structural transitions in the enzyme, revealed by a different heat stability. ATP4- facilitates the process of thermal inactivation by inducing the presence of thermolable forms. On the contrary, ATP-Mg protects the enzyme from thermal inactivation. The protection is significant but less marked than that shown by NADP. Kinetic studies indicate a different type of binding between ATP-Mg and ATP4-. Uncomplexed ATP is a competitive inhibitor vs NADP or G6P and it shows a positive cooperative effect. ATP-Mg is an uncompetitive inhibitor vs G6P and competitive vs NADP and it shows a negative cooperativity. A similar behaviour is also shown by complexed and free CTP and GTP.

Adenosine Triphosphate↗
PubMed1995

Study of the molecular defects in pyruvate kinase deficient patients affected by nonspherocytic hemolytic anemia.

We have examined DNA from fifteen unrelated pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic anemia (HNSHA) for the molecular alterations responsible for the enzyme deficiency. All but 3 of the 30 putative mutations were identified. Fourteen different mutations were found. Nine were missense mutations: 320 T-->C, 823 G-->C, 1276 C-->T, 1378 G-->A, 1484 C-->T, 1529 G-->A, 1654 G-->A, 1675 C-->G; three were nonsense mutations: 603 G-->A, 721 G-->T, 1501 C-->T; one was an insertion at 1574 GGG-->GGGG and the other a three nucleotide in-frame deletion 391-392-393 ATC. Eight of these mutations have not been previously described. We also investigated all of the patients for the C/A polymorphism at nt 1705 and the microsatellite ATT repeat in intron 11. All of the mutations that had previously been reported by us (391-393del, 721T, 1484T, 1529A) were found in the context of the same haplotype as the earlier cases, supporting the concept that each may have a single origin.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1995

Muscle expression of glucose-6-phosphate dehydrogenase deficiency in different variants.

Muscle expression of G6PD deficiency has been investigated in Mediterranean, Seattle-like and A-variants. G6PD activity was detected in samples obtained from biopsies on the quadriceps muscle of seven males and one female. The type of genetic variant was determined by molecular analysis of DNA, extracted from blood samples. All variants showed the enzyme defect in muscle. A statistically significant relationship was found in the activity of G6PD between erythrocytes and muscle of the male subjects (r = 0.968; p = 0.00008). The equation for the best fit line was: Y = 0.390X + 0.198. The results suggest that, for a given variant, the extent of the enzyme defect in muscle may be determined, using this equation, from the G6PD activity of erythrocytes.

Adolescent↗
PubMed1995

Molecular study of pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic anemia.

DNA analysis was performed on 30 unrelated patients with hereditary nonspherocytic hemolytic anemia (HNSHA) who had been found to be pyruvate kinase (PK) deficient by enzyme assay. 19 different mutations were identified among 58 of the 60 alleles at risk. 13 of these were missense mutations that caused single amino acid changes. Included were the following nucleotide substitutions: 401A, 464C, 993A, 1022C, 1076A, 1178G, 1179A, 1373A, 1378A, 1456T, 1484T, 1493A, 1529A. The remaining six mutations were as follows: two nonsense mutations, 721T and 808T; a nucleotide deletion, 307C; a nucleotide insertion, 1089GG; a three nucleotide in frame deletion, 391-392-393 and a deletion of 1149 bp from the PKLR gene that resulted in the loss of exon 11. All the patients were studied for two polymorphic sites, nucleotide (nt) 1705 A/C and a microsatellite in intron 11, to better understand the origin of the mutations. The 1529A mutation, which is the most common mutation in the European population, was found in 25 alleles. With a single exception this mutation was in linkage disequilibrium with both of the polymorphic markers, i.e., found with 1705C and 14 repeats in the microsatellite. This finding is consistent with a single origin of this common mutation. Other mutations occurring more than once were of much lower frequency than the 1529A mutation.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1994

Prenatal diagnosis of pyruvate kinase deficiency.

Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a direct method of PCR amplification and restriction endonuclease analysis, detected mutations in fetus genomic DNA. The second method, using two polymorphic sites linked to the PKRL gene, enabled us to establish which chromosome had been inherited from each parent.

Anemia, Hemolytic, Congenital↗