The spectrum of factor XI deficiency in Italy.

Factor XI (FXI) deficiency is a rare inherited bleeding disorder invariably caused by mutations in the FXI gene. The disorder is rather frequent in Ashkenazi Jews, in whom around 98% of the abnormal alleles is represented by Glu117X and Phe283Leu mutations. A wide heterogeneity of causative mutations has been previously reported in a few FXI deficient patients from Italy. In this article, we enlarge the knowledge on the genetic background of FXI deficiency in Italy. Over 4 years, 22 index cases, eight with severe deficiency and 14 with partial deficiency, have been evaluated. A total of 21 different mutations in 30 disease-associated alleles were identified, 10 of which were novel. Among them, a novel Asp556Gly dysfunctional mutation was also identified. Glu117X was also detected, as previously reported from other patients in Italy, while again Phe283Leu was not identified. A total of 34 heterozygous relatives were also identified. Bleeding tendency was present in very few cases, being inconsistently related to the severity of FXI deficiency in plasma. In conclusion, at variance with other populations, no single major founder effect is present in Italian patients with FXI deficiency.

Reduced von Willebrand factor secretion is associated with loss of Weibel-Palade body formation.

BACKGROUND: von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF) that have different pathophysiologic effect in causing low plasma VWF levels. Type 1 VWD includes quantitative plasma VWF deficiency with normal VWF structure and function. OBJECTIVES: We report three novel type 1 VWF mutations (A1716P, C2190Y and R2663C) located in different VWF domains that are associated with reduced secretion and reduced formation of elongated Weibel-Palade body (WPB)-like granules. METHODS: Transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed and secretion, collagen binding and GpIb binding assessed in comparison with wild-type VWF. Expression was also examined in HEK293 cells that form WPB-like granules when transfected with wild-type VWF. RESULTS: Laboratory results and multimer analysis of plasma VWF was compatible with type 1 VWD. Expression experiments demonstrated slightly reduced VWF synthesis and drastically impaired secretion upon homozygous expression. In HEK293 cells, homozygous expression of A1716P and C2190Y VWF variants failed to form elongated WPB-like granules, while R2663C was capable of WPB-like granules. Heterozygous expression of VWF variants had a negative impact on wild-type VWF with a reduction in elongated WPB-like granules in co-transfected cells. CONCLUSIONS: Our results demonstrate that homozygous and heterozygous quantitative VWF deficiency caused by missense VWF mutations in different VWF domains can be associated with inability to form endothelial WPB-like granules.

Deep intronic variations may cause mild hemophilia A.

BACKGROUND: In about 10% of patients with mild hemophilia A, no candidate gene mutations are apparent after complete gene sequencing. AIM OF THE STUDY: To analyze factor VIII gene (F8) mRNA for mutations in five families with mild hemophilia A with no apparent genomic mutation and a reduced response to desmopressin. RESULTS: In four cases, mRNA studies revealed the presence of an abnormal mRNA transcript in addition to normal F8 mRNA. Sequencing of the abnormal transcripts revealed complex abnormalities, which allowed the identification of three different intronic variations (c.2113+1152delA, c.5587-93C>T and c.5999-277G>A) at the DNA level, absent from 387 normal alleles. By in silico analysis, c.2113+1152delA and c.5587-93C>T were strongly predicted to result in the generation of new splice sites with the introduction of premature termination codons, while c.5999-277G>A was predicted to generate a new protein with 30 additional amino acids. However, these predictions were not homogeneous across the different mutations and programs used. The detrimental effect of two mutations was also confirmed by in vitro expression studies. These changes were also identified in related female carriers and in other mild HA patients not included in the original study. No mRNA abnormality was identified in the remaining patient. CONCLUSIONS: Although rare, deep intronic variations may be responsible for mild hemophilia A where no other F8 mutations have been identified and may be associated with a reduced biologic response to desmopressin. F8 mRNA analysis is a useful tool for the identification of deep intronic variation not detectable by standard DNA sequencing.

Alterations of mRNA processing and stability as a pathogenic mechanism in von Willebrand factor quantitative deficiencies.

INTRODUCTION: von Willebrand disease (VWD) is an inherited bleeding disorder due to a deficiency or abnormality of von Willebrand factor (VWF), associated with heterogeneous phenotypes. While VWD mutations acting at the protein level have been deeply investigated, fewer data are available on genetic defects affecting VWF mRNA. AIM: The aim of this study was to characterize the molecular mechanism underlying VWD in three patients. METHODS: Mutational screening of the patients (P1-3) was accomplished by DNA sequencing of all VWF exons and splicing junctions. Platelet mRNA was analyzed by reverse-transcription (RT)-PCR and real-time RT-PCR. RESULTS: P1 is a compound heterozygote for a c.1534-3C>A transversion in intron 13 and for a nonsense mutation (p.Q77X) in exon 4. P2 is heterozygous for a splicing mutation in intron 9 (c.1109+2T>C). RT-PCR assays on the patient's platelet RNA revealed three mRNA populations: (i) wild type; (ii) lacking exon 9; and (iii) lacking exons 8 and 9. P3 showed a novel homozygous splicing mutation in intron 46 (c.7770+1G>T), producing three different mRNA species: (i) retaining the first 25 bp of intron 46; (ii) skipping exon 46; and (iii) skipping exon 46 while retaining 5 bp of intron 45. Whenever possible, the effect of mutations on the levels of VWF transcripts was analyzed, showing that mRNA variants containing a premature termination codon are downregulated, probably by the nonsense-mediated mRNA decay pathway. CONCLUSIONS: The identification of the genetic basis of VWD in three patients confirmed that mutations leading to null alleles in the VWF gene are associated with allele-specific mRNA degradation.

Homozygous type 2N R854W von Willebrand factor is poorly secreted and causes a severe von Willebrand disease phenotype.

BACKGROUND: von Willebrand disease (VWD) type Normandy (VWD 2N) is caused by mutations at the factor (F)VIII-binding site of von Willebrand factor (VWF), located in the D'and D3 domains on the N-terminus of mature VWF. The R854Q mutation is the most frequent cause of this phenotype. OBJECTIVES: We report the characterization of a homozygous VWD 2N mutation, R854W, detected in a patient with a severe VWD phenotype. METHODS: The plasma VWF phenotype was studied, transient expression of recombinant mutant full-length VWF in 293 EBNA cells was performed, and the results were compared with those obtained with wild-type (WT) VWF. Furthermore, expression was also examined in HEK293 cells, which form Weibel-Palade body-like granules when transfected with WT VWF. RESULTS: The multimer analysis of plasma VWF showed the lack of the typical triplet structure, with the presence of the central band only, and a relative decrease in the high molecular mass multimers. Homozygous expression of recombinant R854W VWF resulted in normal amounts of cellular VWF, but with a severe reduction in secretion into the medium. Severe reductions in FVIII binding to R854W VWF, glycoprotein Ib binding activity and collagen binding of secreted W854 VWF was observed, and reproduced the phenotypic parameters of plasma VWF. In HEK293 cells, homozygous R854W VWF failed to form Weibel-Palade body-like granules. CONCLUSIONS: Our results demonstrate that a homozygous R854W mutation in the D' domain of VWF induces impaired secretion and activity of the protein, thereby explaining the severe phenotype of the patient.

F8 mRNA studies in haemophilia A patients with different splice site mutations.

SUMMARY: Analysis of cDNA is a useful way of investigating splicing mutations and provides more information than using in silico analysis to understand disease pathogenesis better. For understanding the manner in which mutations result in haemophilia A (HA) of different degrees of severity in four index cases with HA and splice site mutations, we performed a detailed analysis of F8 lymphocyte mRNA using a nested PCR-approach. A c.601 +5 G>A change in a mild HA patient produces four transcripts at mRNA level: wild-type, one skipping exon 4, one skipping exons 4 and 5 and one skipping exons 4, 5 and 6, while in silico analysis predicts that the splicing score is not reduced significantly. F8 mRNA of a c.1538 -18 G>A mutation in mild HA lacks the first 36 bases (c.1538_1573del36) of exon 11, resulting in a protein lacking the first 12 amino acids coded for by exon 11, while in silico prediction suggests the creation of a new acceptor splice site with the introduction of 16 bp of intron 10 in the reading frame of exon 11. In keeping with in silico prediction, a c.1443 +1 G>C mutation produces a truncated protein of only 465 amino acids and a c.602 -1 G>A change produces the skipping of exon 5 at mRNA level. Both mutations were identified in severe HA. F8 mRNA analysis is a useful tool for the characterization of the mechanisms by which splice site mutations affect the phenotype, while in silico analysis may not be always reliable.

Molecular and phenotypic determinants of the response to desmopressin in adult patients with mild hemophilia A.

BACKGROUND: The relationship of the biologic response to desmopressin with the F8 mutation and physiological characteristics has been poorly investigated in patients with mild hemophilia A. OBJECTIVES: We prospectively assessed the molecular and phenotypic determinants of the biologic response to desmopressin in a cohort of 50 patients with mild hemophilia A. METHODS: Up to 24 h after desmopressin, blood samples were serially obtained and factor (F)VIII and von Willebrand factor (VWF) measured. The promoter region, all exons and exon-intron boundaries of the F8 gene were screened using denaturing high-performance liquid chromatography (DHPLC). Direct sequencing was done when DHPLC screening was normal. Genomic DNA was also sequenced for exons 18-21, 24 and 27 of VWF. RESULTS: Mean basal FVIII:C was 19 +/- 9 IU dL(-1) (range 6-37) and the median postdesmopressin peak increase was 2.5-fold (range 1.1-7.1). Eleven patients with a cross-reacting material positive (CRM(+)) phenotype had similar basal levels and relative increases of FVIII:C to the remaining patients with low FVIII:Ag. Using multivariate regression, FVIII:C half-life was positively related to basal and peak VWF:Ag levels (P = 0.008) and patient age (P = 0.004). Eleven patients had evidence of reduced FVIII survival. While 27 different gene mutations were identified in 41 patients, nine patients had no detectable mutation. These patients had significantly smaller peaks and smaller relative increase of postdesmopressin FVIII:C (median FVIII:C 26 IU dL(-1) vs. 54 IU dL(-1); P < 0.001; fold 1.8 +/- 0.6 vs. 2.9 +/- 0.8; P = 0.002). CONCLUSIONS: In this cohort of patients with mild hemophilia A, a poor biologic response to desmopressin was frequently associated with the absence of detectable F8 mutations.

Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A.

To provide a National database, 1,410 unrelated hemophilia A (HA) patients were investigated using screening methods denaturing high-performance liquid chromatography (DHPLC), conformational-sensitive gel electrophoresis (CSGE)] and/or direct sequencing. F8 gene mutations were identified in 877 (81%), 146 (82%), and 133 (89%) families with severe, moderate, or mild HA, respectively. Among the 382 different mutations detected, 217 (57%) have not previously been reported in the F8 Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database. Mutations leading to a null allele accounted for 82, 15%, and less than 1% of severe, moderate, or mild HA, respectively. A missense mutation was identified in 16%, 68%, and 81% of severe, moderate, or mild HA, respectively. They included 105 missense mutations (48%), 41 small deletions (19%), 25 splice site mutations (12%), 24 nonsense mutations (11%), 18 insertions (8%), three large deletions (1%), and one deletion plus insertion. Unreported mutations were distributed throughout the F8 gene, as they affected all F8 exons but exon 20. We report a wide spectrum of mutations collected in a large National database. The type of mutation was a strong predictor of the clinical phenotype. This database is expected to considerably improve the genetic counseling and medical care of HA families in Italy.

Molecular characterization of five Italian families with inherited severe factor XIII deficiency.

Factor XIII (FXIII) deficiency is a very rare (1:2 000 000) severe autosomal recessive bleeding disorder, mostly due to mutations in the coagulation FXIII A-subunit gene. We have studied the molecular basis of FXIII deficiency in five unrelated Italian families. The coding region, intron-exon boundaries and 5'- and 3'-untranslated regions of the FXIII gene encoding the A subunit were amplified and directly sequenced. Candidate mutations were identified in all the patients. Three novel mutations occurred in three patients. These include a novel homozygous deletion of two base pairs (bp) in exon 14 (c.2002-2003 DelCT). This deletion causes a frameshift from Leu667 and the formation of a stop codon at amino acid position 681. The second patient presents a novel homozygous (c.2126 G>A) transition in exon 15, predicting a Ser708Asn in Barrel 2 domain. The third patient is compound heterozygote for two missense mutations, a previously reported Arg260His substitution, and a novel transition in exon 4 (c.560 C>T) predicting a Pro186Leu in the core domain. The remaining two patients have two previously reported mutations: a 4-bp homozygous deletion in exon 11 (c.1392-1395 Del AATT), previously reported to occur in the Vicenza Area, and a homozygous nonsense mutation in exon 8 (c.979 C>T) predicting an Arg326X in the core domain. The novel mutations occurred at amino acid residues highly conserved among different species (pig, monkey, mouse and dog) and were not detected in 110 normal alleles. Structural analysis shows that Pro186Leu mutation leads to the replacement of the rigid proline pyrrolidine ring by the larger and more flexible leucine side chain and Ser708Asn may probably disrupt the hydrogen bond with Ala291.

Spectrum of mutations in Albanian patients with haemophilia A: identification of ten novel mutations in the factor VIII gene.

Genetic analysis was carried out in 37 Albanian patients with haemophilia A. The factor VIII intron 22 inversion was detected only in 2/19 (10.5%) apparently unrelated patients with severe haemophilia A, while the intron 1 inversion was absent. A total of 19 different gene mutations were identified. Ten mutations were novel: four null mutations in severe haemophilia A patients (Gln1090X, Cys1832X, 2374delT, 5676insT) and six missense mutations (five in severe haemophilia A) (Ile76Thr, Leu299Pro, Asp525Glu, Cys692Tyr, His1755Leu and Trp1835Cys). None of these novel mutations occurred at CpG hotspots. These results further emphasize the extreme heterogeneity of the molecular basis of haemophilia A. The low prevalence of intron 22 inversion in Albanian patients with severe haemophilia A should be addressed by further studies.