Margherita Scapin

Distinct Promoters Determine Alternative Transcription of gpx-4 into Phospholipid-Hydroperoxide Glutathione Peroxidase Variants

A nuclear variant of phospholipid-hydroperoxide glutathione peroxidase (PHGPx, GPx-4) was considered to be derived from alternative pre-mRNA splicing in testis and to regulate sperm maturation. The genomic sequence of rat gpx-4 was established and investigated in respect to expression into the cytosolic, mitochondrial, and nuclear forms of PHGPx. In silico analysis suggested the presence of two distinct promoter regions, the upstream one leading to transcripts translating into cPHGPx or mPHGPx and the downstream one yielding nPHGPx. The promoter activity of both regions was verified by luciferase-based reporter constructs in A7r5 and H9c2 cells. The data reveal that the formation of nPHGPx is due to alternative transcription and not to alternative splicing. Transcripts encoding nPHGPx were most abundant in testis although not restricted to this organ. This observation points to a general role of the nuclear PHGPx variant in regulating cell division.

Genetic Variations of gpx-4 and Male Infertility in Humans

Abstract Phospholipid hydroperoxide glutathione peroxidase (PHGPx), the product of gpx-4, is the major selenoprotein in sperm and is considered essential for fertilization because of its multiple roles in spermatogenesis, such as hydroperoxide detoxification, formation of the mitochondrial capsule, and chromatin condensation. Genomic DNA sequences of 3.148 kilobases covering the whole gpx-4 and its flanking regions were amplified from 63 men using the polymerase chain reaction and were analyzed for polymorphisms by direct sequencing. A total of 23 variant sites were detected; 2 were present only in control men (proven fathers; n = 21) and 10 were common to fertile controls and infertile patients (n = 42). A further 11 variant sites were seen in five of the infertile men only. Four of the gpx-4 variants were considered irrelevant to GPx-4-related fertility problems because they occurred homozygously in controls. The majority of the remaining variant sites are also of questionable relevance because they are located in introns or, as third base exchanges, do not affect the protein sequence. However, one of the exon variations leads to an Ala93-Thr exchange that reduces activity in a porcine GPx-4 homologue. Two detected promoter variations were shown by reporter gene constructs to affect transcription in somatic cell lines. These results indicate that gpx-4 polymorphism cannot generally account for the correlation of PHGPx content of sperm and fertility-related parameters, but further examination of this gene as a potential cause of infertility in particular cases is warranted.

Novel point mutation in a leucine-rich repeat of the GPIbα chain of the platelet von Willebrand factor receptor, GPIb/IX/V, resulting in an inherited dominant form of Bernard-Soulier syndrome affecting two unrelated families: the N41H variant

This reports describes a new variant of heterozygous Bernard-Soulier syndrome with autosomal dominant inheritance. In Italy, a significant proportion of patients with autosomal dominant inheritance of macrothrombocytopenia have been recognized as having heterozygous Bernard-Soulier syndrome carrying the Bolzano-type defect. This condition prompted a systematic review of our out-patients with chronic isolated macrothrombocytopenia. We recognized that the affected members of two unrelated families represented a new variant of heterozygous Bernard-Soulier Syndrome with autosomal dominant inheritance. Sequencing analysis of the GPIbα gene revealed a novel heterozygous mutation, A169C, resulting in an N41H substitution in the protein. This aminoacid belongs to the first leucine-rich repeat of the chain. The molecular modeling suggests that the replacement of the N41 with a histidine (N41H) drastically disturbs the structure of the first portion of GPIbα N-terminal, directly involved in von Willebrand factor binding. As a consequence, platelet aggregation to 1.2 mg/mL of ristocetin is slightly impaired and flow cytometry reveals a reduced binding of monoclonals directed against N-terminal epitopes of the GPIbα.

Arterial and venous thrombosis in patients with von Willebrand’s disease: A critical review of the literature

All patients with von Willebrand’s disease (vWD) who showed an arterial or venous thrombosis and were reported in the literature have been evaluated. 11 patients had arterial thrombosis while 19 had venous thrombosis for a total of 30 cases. 9 out the 11 cases with arterial thrombosis had myocardial infarction. Two had cerebral thrombosis. Associated risk factors for arterial thrombosis were available only for three patients who showed, respectively, smoking and dyslipidemia (2 cases) and smoking and intravenous desmopressin infusion (1 case). The majority of patients with venous thrombosis showed DVT with or without PE. Four patients presented with apparently isolated PE. In two instances thrombosis occurred in unusual sites (central retinal vein and portal vein, respectively). Several associated risk factors were present, mainly: infusion of FVIII or FVIII + vWF concentrates in 7 cases; surgery in 8 cases, pregnancy in 1, desmopressin infusion in 1, variable coagulation defects or polymorphisms in 5. More than one of these associated conditions were present in a few patients. The majority of vWD patients who showed thrombotic phenomena were type I patient, but in 6 cases were also type 3. The type of defect was not reported in 6 patients. As a conclusion of this review it seems safe to assume that both arterial and venous thrombosis appear rare in vWD. This is confirmed by the fact that arterial or venous thrombosis appears slightly more frequent in hemophilia A and B.

Heparin-Induced Thrombocytopenia in Patients with Philadelphia-Negative Myeloproliferative Disorders and Unusual Splanchnic or Cerebral Vein Thrombosis

Background: Philadelphia-negative myeloproliferative disorders (Ph-MPD) are common causes of unusual splanchnic or cerebral vein thrombosis, which is treated with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). Heparin-induced thrombocytopenia (HIT) is a dangerous potential complication of this therapy, but it has rarely been reported in Ph-MPD. Patients and Methods: We retrospectively reviewed clinical records of 29 patients with Ph-MPD who have been treated with UFH or LMWH for unusual splanchnic or cerebral vein thrombosis (3 cerebral sinus, 6 portal and 20 hepatic vein). The goal of the study was to determine the occurrence of new thrombotic events during heparin therapy secondary to HIT (HITT). Results: During heparin therapy, 5 out of the 29 patients (17%) developed a new thrombotic episode (pulmonary embolism) with a high clinical probability of HIT based on the 4 T’s score even though not all the patients developed ‘true’ thrombocytopenia. A diagnosis of HIT was established in 2 patients (6.8%) through the presence of heparin-related antibodies. Conclusions: Ph-MPD patients on heparin warrant careful monitoring and HIT has to be suspected whenever platelet counts drop or a new thrombosis is detectable.

Congenital FX deficiency combined with other clotting defects or with other abnormalities: a critical evaluation of the literature

Summary.  The presence of more than one congenital clotting defect in a given patient is a rare event but not an exceptional one. Combined defects of factor X (FX) are very rare because congenital isolated FX deficiency is by itself very rare. A perusal of personal files and of the literature has yielded 12 families with FX deficiency in which an association with another clotting factor deficiency was found. The associated defects were factor VII (FVII) or factor VIII (FVIII) or factor XII (FXII) deficiency. By far the most frequently associated was with FVII. Two forms of this association were found. In the first form there is casual association of both FVII and FX deficiency in the proband with independent recessive segregation of the two defects in other family members. The second form is because of abnormalities in chromosome 13 (deletions, translocations and so on) involving both FX and FVII genes. These genes are known to be very close and located on the long arm of chromosome 13 at about 13q34. In this form the hereditary pattern is autosomal dominant. Isolated FX deficiency and, more frequently, combined FX + FVII deficiency appear also associated with coagulation‐unrelated abnormalities (carotid body tumours, mitral valve prolapse, atrial septal defect, ventricular septal defect, thrombocytopenia absent radius (TAR) syndrome, mental retardation, microcephaly and cleft palate). Diagnosis of a combined clotting defect could be difficult on the basis of global tests. For example, both isolated FX deficiency and combined FX + FVII deficiency yield a prolongation of basal PTT and PT. Only specific assays could allow one to reach the correct diagnosis. In cases of casual association with other defects, it is also important to study family members, as the two defects should segregate independently.

Src tyrosine kinase preactivation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombo-hemorrhagic complications. The acquired somatic Janus kinase 2 (JAK2) V617F mutation is present in the majority of PV and ET patients. Because aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the V617F mutation. In addition, Src was inactive in all resting platelets, but in the pathological specimens it was present in a preactivated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by Src homology-2 domain-containing protein Tyr-phosphatase-2 (SHP-2), whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca(2+)](c) increase, and aggregation in pathological platelets compared with controls. Thrombin-induced Src activation preceded JAK2 activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src kinase preactivation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

Genetic study in patients with factor XII deficiency : a report of three new mutations exon 13 (Q501STOP), exon 14 (P547L) and -13C>T promoter region in three compound heterozygotes

A group of 29 patients with congenital factor XII (FXII) deficiency belonging to nine distinct families have been investigated. All were cases of true deficiency in the sense that there was no discrepancy between FXII activity and FXII antigen. From a clotting point of view, 11 patients appeared homozygous, as both FXII activity and antigen were very low (≤1% and traces of antigen). In other words, they were cases with no cross-reactivity material. In the heterozygotes, FXII activity and antigen were about 50% of normal in all cases. The molecular studies revealed that seven patients were real homozygotes for the mutation −8G>C in the promoter region confirming the conclusions reported by coagulation tests. On the contrary, the remaining patients with a homozygote-like phenotype were instead found to be compound heterozygous for two distinct mutations. Three of these mutations were new mutations, namely the combination of −8G to C with 501Q to T (exon 13), 547P to L (exon 14) and −13C to T in the promoter, respectively. The remaining mutations seen were not new. It is interesting that all compound heterozygotes showed a clotting and immunological pattern similar to that shown by homozygotes, namely very low FXII activity and antigen. The new mutations were not present in the group of 98 normal persons of both sexes with the same geographical background. The wide diffusion of the −8G>C mutation in this group of patients coming from a limited geographical area suggests a founder effect. The significance and importance of genetic analysis in addition to clotting and immunological studies in FXII deficiency is emphasized.

JAK2V617F mutation is common in old patients with polycythemia vera and essential thrombocythemia

Background: JAK2V617F mutation occurs in 90% of polycythemia vera (PV) and in 50% of essential thrombocythemia (ET) patients. Materials and methods: 253 consecutive patients affected by myeloproliferative disorders (MPD, 121 PV, 132 ET) were evaluated and stratified in 4 age groups: 18–39, 40–59, 60–75 and over 75 years (>75). The JAK2V617F mutation was searched and its allele burden was evaluated. Results: The percentage of mutated patients increased progressively with age mainly in patients >75 (p=0.0015 vs 18–39, p=0.0021 vs 40–59 and p=0.012 vs 60–75). We also found a progressive increase in allele burden with age (R2=0.042). Thrombotic events were more common in patients carrying the mutation in comparison with wild type (WT) (p=0.006, coefficient risk 1.94). No differences in the percentage of patients carrying the JAK2V617F mutation were found, in spite of different follow-up durations (<5 yrs, 5–10 yrs, 10–15 yrs, >15 yrs). The JAK2V617F allele burden was similar in patients with (57±31%) and without (45±26%) long-term hydroxyurea treatment. Conclusions: JAK2V617F mutation is more common in old than in young patients with MPD. Older patients have an higher allele burden.

A case of Bernard-Soulier Syndrome due to a homozygous four bases deletion (TGAG) of GPIbalpha gene: lack of GPIbalpha but absence of bleeding.

More than 20 DNA mutations with different inheritance pattern have been described in patients with Bernard-Soulier Syndrome (BSS), leading to abnormal or absent synthesis and/or expression of GPIbα. Clinical phenotype shows considerable variation between individuals, such as bleeding, platelet count and the percentage of large platelets. We describe in a BSS patient the first case of homozygous four bases deletion (TGAG) in the gpIbα gene coding sequence, leading to a premature stop codon. In the propositus, blood smears revealed giant platelets (30 × 109 platelets/L), and platelet agglutination to ristocetin was absent. Propositus’ parents are consanguineous. His father and paternal grandmother showed a mild thrombocytopenia (108 × 109/L and 120 × 109/L platelets respectively) while mothers and sisters referred normal platelet counts. The surface expression of GPIbα was practically undetectable by flow-cytometry and western blot in the patient and was reduced in the father. Probands DNA analysis revealed a homozygous four-base-pair deletion (TGAG), starting from the last base of the codon for Ser39, leading to a coding frame shift with a new termination codon after 11 novel amino acids. The same mutation was seen in heterozygosis in both parents. This is the first report of GPIbα TGAG deletion in homozygous state even if the defect has already been described in a case of compound heterozygosis. Surprisingly, the propositus does not report any spontaneous bleeding tendency.